JP4385720B2 - Transformed cells with enhanced sensitivity to antimicrobial active substances and use thereof - Google Patents

Description

  The present invention relates to a transformed cell having enhanced sensitivity to an antibacterial active substance and use thereof.

When fungicides containing dicarboximide antibacterial active substance, aromatic hydrocarbon antibacterial active substance or phenylpyrrole antibacterial active substance as an active ingredient act on certain phytopathogenic fungi, the fungus is high. It is known that intracellular glycerol synthesis increases as in the case of osmotic stress, and the intracellular osmotic pressure cannot be controlled and die. Due to its action against phytopathogenic fungi, a protein of an information transmission system related to osmotic pressure control was expected as a target protein of an antibacterial active substance contained as an active ingredient in these fungicides.
A mutant os-1 having a sensitivity to osmotic pressure was reported in Neurospora crassa which is sensitive to the above-mentioned antibacterial active substances. This mutant os-1 exhibits resistance to the above antibacterial active substances. By analyzing the mutant strain, the OS-1 gene, which is an osmotic pressure sensitive histidine kinase gene, was isolated as a causative gene. A protein having an amino acid sequence encoded by the base sequence of this OS-1 gene has a structure of histidine kinase of a two-component control system and a polypeptide consisting of about 90 amino acids having amino acid sequence homology with each other six times. It was a protein having a characteristic sequence region that repeatedly exists (hereinafter also referred to as a repeated sequence region) (for example, Patent Document 1, Non-Patent Document 1, Non-Patent Document 2, Non-Patent Document 3, (See Patent Document 5 and Non-Patent Document 6.) Genes having homology to the OS-1 gene include plant pathogenic fungi such as Botryotinia fuckeliana, rice blast fungus (Magnaporthe grisea), and pea root rot fungus (Fusarium solani). The nucleotide sequence and amino acid sequence are also disclosed. It is known that homologous genes for these OS-1 genes exist specifically in filamentous fungi among eukaryotic organisms (see, for example, Non-Patent Document 4, Non-Patent Document 7, and Non-Patent Document 8). .)

US Pat. No. 5,939,306 GneneBank accession U50263, U53189, AAB03698, AAB01979 Alex, A.L. et al., Proc. Natl. Acad. Sci. USA 93: 3416-3421 Schumacher, M.M. et al., Current Microbiology 34: 340-347 GneneBank accession AF396827, AF435964, AAL37947, AAL30826 Oshima, M. et al., Phytopathology 92 (1): 75-80 Fujimura, M. et al., J. Pesticide Sci. 25: 31-36 Fujimura, M. et al., Pesticide Biochem. Physiol. 67: 125-133 GneneBank accession AB041647, BAB40497

As described above, the OS-1 protein and the protein group having homology to the protein are proteins specific to filamentous fungi and are important proteins related to osmotic pressure control. A new antibacterial activity assay method using the OS-1 gene and a gene having homology to the gene based on the assumption, a method for efficiently selecting an antibacterial active substance, etc. Tried to develop.

Under such circumstances, as a result of intensive studies, the inventor has found a transformed cell having enhanced sensitivity to an antibacterial active substance, a novel antibacterial activity assay method using this transformed cell, and this trait The present inventors have found a method for selecting an antibacterial active substance using transformed cells and have reached the present invention.

That is, the present invention
1. A gene comprising a base sequence encoding an amino acid sequence of an osmotic pressure sensitive histidine kinase having no transmembrane region is introduced into a cell deficient in at least one hybrid sensor kinase in a functional form. Transformed cells;
2. A hybrid sensor in which a gene comprising a base sequence encoding the amino acid sequence of an osmotic pressure sensitive histidine kinase having no transmembrane region to be introduced is deficient in at least one hybrid sensor kinase. 2. The transformed cell according to item 1 above, which is a gene complementary to a kinase function;
3. The transformed cell according to item 1 above, wherein the cell is a microorganism;
4). The transformed cell according to item 1, wherein the microorganism is budding yeast;
5. The gene comprising the base sequence encoding the amino acid sequence of osmotic pressure sensitive histidine kinase that does not have a transmembrane region is any of dicarboximide antibacterial active substance, aromatic hydrocarbon antibacterial active substance, and phenylpyrrole antibacterial active substance. 2. The histidine kinase having a resistance mutation against mosquito and a gene comprising a base sequence encoding the amino acid sequence of histidine kinase having no transmembrane region and being sensitive to osmotic pressure, characterized in that Transformed cells;
6). 2. The transformed cell according to item 1 above, wherein the osmosensitive histidine kinase having no transmembrane region is an osmotic sensitive histidine kinase having no transmembrane region, which is derived from a phytopathogenic filamentous fungus;
7). Osmotic-sensitive histidine kinase that does not have a transmembrane region is derived from gray mold fungi, rice blast fungus, spinach wilt fungus, wheat leaf blight fungus, rice blight fungus or tomato blight fungus The transformed cell according to the above item 1, which is an osmotic pressure sensitive histidine kinase having no transmembrane region;
8). 1) The amino acid sequence of an osmotic pressure sensitive histidine kinase having no transmembrane region is the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 16, SEQ ID NO: 41, SEQ ID NO: 55 or SEQ ID NO: 68 8. Transformed cells as described; The base sequence encoding the amino acid sequence of an osmotic pressure sensitive histidine kinase that does not have a transmembrane region is a base sequence represented by SEQ ID NO: 2, SEQ ID NO: 17, SEQ ID NO: 42, SEQ ID NO: 56 or SEQ ID NO: 69. The transformed cell according to item 1 above, characterized by:
10. A method for assaying antibacterial activity of a substance,
A first step of culturing the transformed cell according to item 1 in the presence of a test substance;
A second step of measuring an intracellular signal transduction amount from an osmotic pressure sensitive histidine kinase not having a transmembrane region expressed in the transformed cell cultured in the first step or an index value correlated therewith,
A third step of evaluating the antibacterial activity of the test substance based on the difference between the intracellular signal transmission amount measured in the second step or an index value correlated therewith and a control,
An assay method characterized by comprising:
11. 11. The assay method according to item 10 above, wherein the intracellular signal transduction amount from an osmotic pressure sensitive histidine kinase not having a transmembrane region or an index value correlated therewith is the growth amount of the transformed cell.
12 A method for searching for an antibacterial active substance, which comprises selecting an antibacterial active substance based on the antibacterial activity evaluated by the assay method of the preceding item 10;
13. 13. An antibacterial active substance selected by the searching method according to item 12 above.
14 An osmosensitive histidine kinase derived from a phytopathogenic fungus and having no transmembrane region;
15. A polynucleotide having a base sequence encoding an amino acid sequence of an osmotic pressure sensitive histidine kinase which is derived from a phytopathogenic fungus and does not have a transmembrane region;
16. Histidine kinase <amino acid sequence> having any of the following amino acid sequences
(A) the amino acid sequence represented by SEQ ID NO: 41, SEQ ID NO: 55 or SEQ ID NO: 68,
(B) An osmotic pressure that is an amino acid sequence in which one or more amino acids are deleted, added, or substituted in the amino acid sequence represented by SEQ ID NO: 41, SEQ ID NO: 55, or SEQ ID NO: 68, and has no transmembrane region. The amino acid sequence of the sensitive histidine kinase,
(C) the amino acid sequence of an osmotic pressure sensitive histidine kinase which is an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown by SEQ ID NO: 41, SEQ ID NO: 55 or SEQ ID NO: 68 and which does not have a transmembrane region ;
17. Histidine kinase gene <amino acid sequence> having a base sequence encoding any of the following amino acid sequences
(A) the amino acid sequence represented by SEQ ID NO: 41, SEQ ID NO: 55 or SEQ ID NO: 68,
(B) An osmotic pressure that is an amino acid sequence in which one or more amino acids are deleted, added, or substituted in the amino acid sequence represented by SEQ ID NO: 41, SEQ ID NO: 55, or SEQ ID NO: 68, and has no transmembrane region. The amino acid sequence of the sensitive histidine kinase,
(C) the amino acid sequence of an osmotic pressure sensitive histidine kinase which is an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown by SEQ ID NO: 41, SEQ ID NO: 55 or SEQ ID NO: 68 and which does not have a transmembrane region ;
18. A polynucleotide having the base sequence represented by SEQ ID NO: 42, SEQ ID NO: 56, or SEQ ID NO: 69;
19. A method for obtaining a polynucleotide having a base sequence encoding an amino acid sequence of an osmotic pressure sensitive histidine kinase which is derived from a phytopathogenic filamentous fungus and does not have a transmembrane region. SEQ ID NOS: 30 to 40, 52, 53, 64 , 65, 85, and 86, a step of amplifying a desired polynucleotide by PCR using an oligonucleotide having a base sequence represented by any of the primers, and a step of recovering the amplified desired polynucleotide An acquisition method characterized by comprising:
20. An oligonucleotide having a base sequence represented by any of SEQ ID NOs: 30 to 40, 52, 53, 64, 65, 85, and 86;
Etc. are provided.

  According to the present invention, it is possible to provide a transformed cell with enhanced sensitivity to an antibacterial active substance, and furthermore, an antibacterial activity test method for a test substance using the transformed cell, a method for searching for an antibacterial active substance using the method, etc. Was made available.

Hereinafter, the present invention will be described in detail.
“A gene having a base sequence encoding the amino acid sequence of an osmotic pressure sensitive histidine kinase having no transmembrane region is introduced into a cell deficient in at least one hybrid sensor kinase in a functional form. The “transformed cell characterized” encodes the amino acid sequence of “osmotically sensitive histidine kinase that does not have a transmembrane region” in the “cell deficient in at least one or more hybrid sensor kinases” that is a host cell. It is obtained by introducing a gene consisting of a base sequence in a functional form. Here, “introducing a gene in a functional form” means introducing the gene so as to complement the function of the deficient hybrid sensor kinase in the cell, in other words, by deficient in the hybrid sensor kinase. This means that the gene is introduced in such a way that the induced phenotypic change of the cell can be restored to the phenotype before the defect. Specifically, for example, in the case of budding yeast (for example, Saccharomyces cerevisiae), if a hybrid sensor kinase SLN1 gene (hereinafter also referred to as SLN1 gene) is deficient, it grows under normal growth conditions. Although it indicates a trait that is impossible, it means that a trait that can be grown is exhibited by introducing the SLN1 gene isolated from Saccharomyces cerevisiae into a cell line deficient in this SLN1 gene. The “cell deficient in at least one or more hybrid sensor kinases” can be obtained by deleting at least one or more hybrid sensor kinases inherent in the host cell. First, the hybrid sensor kinase will be described below.

(Two-component control system and hybrid sensor kinase)
The two-component regulatory system is an information transmission system widely used in prokaryotic organisms, and basically consists of two proteins called sensors and regulators. is called. In a typical two-component control system, the sensor protein is composed of an input region and a histidine kinase region, and the regulator protein is composed of a receiver region and an output region. When the input region senses an external stimulus, a histidine residue in an amino acid sequence that is well conserved among species of the histidine kinase region is phosphorylated or dephosphorylated. Here, phosphorylation of a histidine residue is an autophosphorylation reaction using ATP as a substrate. This phosphate group is transferred to an aspartic acid residue in the amino acid sequence that is well conserved among species in the receiver region of the regulator protein, and the presence or absence of phosphorylation of this aspartic acid residue indicates the activity of the output region in the regulator protein. Is adjusted. In prokaryotic organisms, there are exceptions, but the output region is often a transcriptional regulator, and the regulator protein directly regulates gene expression via the aforementioned phosphoryl group transfer in response to stimuli sensed by the sensor protein. Control.
The sensor protein may have a slightly more complicated structure than the typical structure described above. For example, in addition to a structure consisting of an input region and a histidine kinase region, there may be a receiver region continuously found in the regulator protein on the C-terminal side. In this case, the transfer mode of the phosphate group is also complicated, and it is known that the phosphate transfer from the sensor protein to the regulator protein called the response regulator passes through the mediator protein having the transmitter region called the phosphor transmitter. ing. That is, when a stimulus is detected in the input region of the sensor protein, from the histidine residue of the histidine kinase region in the molecule to the aspartic acid residue of the receiver region in the molecule, then to the histidine residue of the phosphotransmitter. Finally, a signal transduction of the phosphate group is made to the aspartic acid residue in the receiver region of the response regulator. Thus, three proteins may be involved in the two-component control system. A sensor protein that is involved in the signal transduction of the phosphate group transfer consisting of these three proteins and has the above structural features is called “hybrid sensor kinase”. Hybrid sensor kinases exist not only in prokaryotic organisms but also in microorganisms or plants that are eukaryotic organisms such as yeast, and are involved in responses to various stimuli and stress.
Here, the input region of the hybrid sensor kinase is a region existing on the N-terminal side of the histidine kinase region, and in many cases has a transmembrane region. This transmembrane region is shown by structure prediction analysis using transmembrane region prediction software such as TMpred program (K. Hofmann & W. Stoffel, Biol. Chem. Hoppe-Seyler, 374, 166 (1993)). It is. The above program is generally available from, for example, http://www.ch.embnet.org/software/TMPRED_form.html. Alternatively, the histidine kinase region of the hybrid sensor kinase is a region present on the C-terminal side of the input region, for example, Parkinson, JS & Kofoid, EC (1989) Annual Review of Genetics 23: 311-336, Stock, JB et.al. (1989) Microbiological Reviews 53 (4): 450-490, which is a region characterized by having five conserved motifs common to common histidine kinases. In the hybrid sensor kinase SLN1, it is the region from the 556th amino acid to the 908th amino acid. The receiver region of the hybrid sensor kinase is a region present on the C-terminal side of the histidine kinase region, for example, Parkinson, JS & Kofoid, EC Annual Review of Genetics 23: 311-336 (1989), Stock, JB et al. (1989) Microbiological Reviews 53 (4): 450-490, a region characterized by having three conserved motifs common to common histidine kinases, for example, hybrids of budding yeast In sensor kinase SLN1, it is the region from the 1088th amino acid to the 1197th amino acid.
In the case of complicated communication of information after the response regulator, in addition to the simple system in which the output region of the regulator is a transcriptional regulator as described above, the MAP kinase cascade related to various controls in the cell is used. It is also known that a signal is transmitted to a transcriptional regulatory factor that controls gene expression.
Hybrid sensor kinases exist not only in prokaryotic organisms but also in microorganisms or plants that are eukaryotic organisms such as yeast, and are involved in responses to various stimuli and stress.
A specific example of hybrid sensor kinase and information transmission involving hybrid sensor kinase will be described below.

(Hybrid sensor kinase of budding yeast)
Budding yeast (Saccharomyces cerevisiae) uses the hybrid sensor kinase SLN1 for signaling related to osmotic pressure control. This SLN1 is the only histidine kinase in budding yeast. SLN1 is an osmotic pressure sensitive histidine kinase having a transmembrane region in the input region, and is known to transmit a phosphotransferase signal to the response regulator SSK1 via the phosphotransmitter YPD1. Downstream of this signal transduction is a MAP kinase cascade consisting of three kinases SSK2 (MAPKKK), PBS2 (MAPKK) and HOG1 (MAPK), which controls gene expression related to osmotic adaptation such as glycerol biosynthesis. . The output region of the response regulator SSK1 has SSK2 phosphorylation activity. SSK1 is negatively regulated by phosphorylating the aspartic acid residue in the receiver region, thereby inhibiting the phosphorylation activity in the output region. Specifically, under normal osmotic pressure, the histidine residue of the histidine kinase region of SLN1 is autophosphorylated, and this phosphate group is transferred to the aspartate residue of the receiver region in the same molecule, and then to the histidine residue of YPD1. And finally to the aspartic acid residue in the receiver region of SSK1. Phosphorylation of the aspartic acid residue in the SSK1 receiver region suppresses the phosphorylation activity of the SSK1 output region, and phosphorylation of the MAP kinase cascade consisting of SSK2, PBS2, and HOG1 is not performed. Furthermore, gene expression related to osmotic pressure adaptation such as glycerol biosynthesis is not induced. On the other hand, under high osmotic pressure conditions, autophosphorylation of histidine residues in the histidine kinase region in SLN1 stops, so the MAP kinase cascade consisting of SSK2, PBS2 and HOG1 is activated, and osmotic pressure adaptation such as glycerol biosynthesis It is known that the gene expression related to is induced (Maeda, T. et.al. (1994) Nature 369: 242-245).

(Hybrid sensor kinase of fission yeast)
In fission yeast (Scchizosaccharomyces pombe), three types of hybrid sensor kinases PHK1 (MAK2), PHK2 (MAK3) and PHK3 (MAK1) are involved in cell cycle control (transition from G2 phase to M phase) and oxidative stress response. ing. There is no histidine kinase other than PHK1, PHK2, and PHK3 in fission yeast. PHK1 and PHK2 are oxidative stress-responsive histidine kinases such as hydrogen peroxide (Buck, V. et. El. Mol. Biol. Cell 12: 407-419). It is known that the three types of hybrid sensor kinases PHK1, PHK2, and PHK3 transmit a phosphoryl group transfer signal to the response regulator MCS4 via the phosphor transmitter SPY1 (MPR1). Downstream of this signal transduction is a MAP kinase cascade consisting of three kinases, WAK1 (MAPKKK), WIS1 (MAPKK), and STY1 (MAPK), which controls gene expression related to cell cycle control and oxidative stress response. . The output region of the response regulator MCS4 has WAK1 phosphorylation activity. MCS4 is negatively regulated by phosphorylation of the aspartic acid residue in the receiver region, thereby inhibiting phosphorylation activity in the output region. Specifically, under normal conditions, the histidine residues in the histidine kinase region of PHK1 to 3 are autophosphorylated, and this phosphate group is transferred to the aspartate residue in the receiver region in the same molecule, and then to the histidine residue in SPY. And finally to the aspartic acid residue in the MCS4 receiver region. Phosphorylation of the aspartate residue in the MCS4 receiver region suppresses the phosphorylation activity of the output region of MCS4, and phosphorylation of the MAP kinase cascade consisting of WAK1, WIS1, and STY1 is not performed Furthermore, gene expression related to cell cycle control and stress response is not induced. On the other hand, under stress conditions, autophosphorylation of histidine residues in the histidine kinase region is stopped in PHK1-3, activating the MAP kinase cascade consisting of WAK1, WIS1, and STY1, thereby regulating cell cycle and oxidative stress response Gene expression related to is induced. As a result, a phenotype is observed in which the cell cycle of the fission yeast is promoted from the G2 phase to the M phase, and the cell length during division is significantly shorter than usual (Aoyama, K. et.al. (2001 Boisci. Biotechnol. Biochem. 65: 2347-2352).

(Bacterial hybrid sensor kinase)
In Escherichia coli, a prokaryotic organism, the hybrid sensor kinase RcsC is involved in regulating the expression of the cps operon involved in capsular polysaccharide synthesis. RcsC is a histidine kinase having a transmembrane region, and is known to transmit a phosphotransferase signal to the response regulator RcsB via the phosphotransmitter YojN. The output region of RcsB has the transcriptional control activity of the cps operon. Specifically, under normal conditions, the histidine residue of the histidine kinase region of RcsC is autophosphorylated, and this phosphate group is transferred to the aspartate residue of the receiver region in the same molecule, and then to the histidine residue of YojN. Finally, it is transferred to the aspartic acid residue in the receiver region of RcsB. By phosphorylating the aspartic acid residue in the RcsB receiver region, cps operon transcriptional activity in the RcsB output region is suppressed, and the gene expression involved in the biosynthesis of capsular polysaccharide is not induced. On the other hand, under high osmotic pressure conditions, autophosphorylation of histidine residues in the histidine kinase region is stopped in RcsC, and the cps operon transcriptional activity in the output region of RcsB is activated, which is involved in the biosynthesis of capsular polysaccharide. Gene expression is induced (Clarke, DJ et.al. (2002) J. Bactriol. 184: 1204-1208).
The bioluminescent marine microorganism Vibrio harveyi fluoresces by luciferase according to its cell density. Hybrid sensor kinases LuxN and LuxQ are involved in the regulation of gene expression related to cell density-responsive bioluminescence. LuxN and LuxQ are histidine kinases having a transmembrane region. V. harveyi produces and secretes two substances (AI-1, AI-2) called Autoinducer in order to sense its own cell density, LuxN senses AI-1, and AI-2 LuxQ Information is transmitted by sensing. LuxN and LuxQ are known to transmit a phosphoryl group transfer signal to the response regulator LuxO via the phosphor transmitter LuxU. LuxO output region has transcriptional control activity of the luciferase operon. Specifically, using LuxN as an example, when the cell density is low, the surrounding histidine residue in the histidine kinase region of LuxN is autophosphorylated because the surrounding AI-1 is low and is not detected in the input region of LuxN. This phosphate group is transferred to the aspartic acid residue of the receiver region in the same molecule, then to the histidine residue of LuxU, and finally to the aspartic acid residue of the receiver region of LuxO. Phosphorylation of the aspartic acid residue in the LuxO receiver region suppresses the luciferase operon transcriptional activity in the LuxO output region and does not induce gene expression related to bioluminescence. On the other hand, under high cell density conditions, AI-1 is abundant, and this is detected by the input region of LuxN. Therefore, autophosphorylation of histidine residues in the histidine kinase region stops in LuxN, and luciferase in the output region of LuxO Operon transcriptional activity is activated and bioluminescence is induced (Freeman, JAet.al. (2000) Mol. Microbiol. 35: 139-149).

(Plant hybrid sensor kinase)
In the higher plant Arabidopsis thaliana, the plant hormone cytokinin receptor proteins CRE1, AHK2 and AHK3 are hybrid sensor kinases. Receptor proteins CRE1, AHK2 and AHK3 are all cytokinin-sensitive histidine kinases having a transmembrane region (Inoue, T. et.al. (2001) Nature 409: 1060-1063). CRE1 is known to transmit a phosphoryl group transfer signal to response regulators ARR1, ARR2, and ARR10 via phosphor transmitters AHP1 and AHP2. The output regions of ARR1, ARR2, and ARR10 are considered to have the transcriptional control activity of cytokinin-inducible genes ARR4-7. Specifically, in the presence of cytokinin, the histidine residue in the histidine kinase region of CRE1 is autophosphorylated, and this phosphate group becomes an aspartate residue in the receiver region in the same molecule, and then histidine in AHP1 and AHP2. To the residue and finally to the aspartic acid residue in the receiver region of ARR1, ARR2 and ARR10. Phosphorylation of aspartic acid residues in the receiver regions of ARR1, ARR2, and ARR10 promotes gene transcription activity in the output regions of ARR1, ARR2, and ARR10 and induces expression of cytokinin-responsive genes ARR4-7 (Hwang, I. & Sheen J. (2001) Nature 413: 383-389).

(Cells deficient in at least one hybrid sensor kinase)
The “cell deficient in at least one or more hybrid sensor kinases” means a cell in which the function of at least one or more hybrid sensor kinases inherent in the cells is lost. Examples of such cells include, for example, cells in which production of at least one or more hybrid sensor kinases inherent in the cells is deleted, suppressed or inhibited, at least one or more hybrid sensor kinases inherent in the cells. Examples thereof include cells whose activity is deleted, suppressed or inhibited. More specific examples include budding yeast lacking SLN1, fission yeast lacking all three of PHK1, PHK2 and PHK3, Escherichia coli lacking RcsC, V. harveyi lacking LuxN, Arabidopsis lacking CRE1, etc. Can be mentioned.
To prepare “cells deficient in at least one or more hybrid sensor kinases”, for example, Saccharomyces cerevisiae TM182 strain deficient in SLN1 is described in Maeda, T. et.al. (1994) Nature 369: 242-245. The fission yeast strain KI011 lacking all three of PHK1, PHK2, and PHK3 can be used as described in Aoyama, K. et.al. (2001) Boisci. Biotechnol. Biochem. 65: 2347-2352. The method can be used. In addition, E. coli SRC122 lacking RcsC can be prepared by the method described in Suzuki, T., et.al. (2001) Plant Cell Physiol. 42: 107-113, and V. harveyi BNL63 lacking LuxN is Freeman. , JA et.al. (2000) Mol. Micobiol. 35: 139-149. Furthermore, Arabidopsis lacking CRE1 is, for example, responsive to cytocanin from a clone obtained by mutagen treatment of Arabidopsis according to the method described in Inoue, T. et.al. (2001) Nature 409: 1060-1063. CRE1 genomic gene fragments are selected by PCR using primers synthesized based on the base sequence of the CRE1 genomic gene listed in Genebank accession AB049934 using the selected clone's genomic DNA as a template. By amplifying and confirming the nucleotide sequence, a defective clone that cannot express CRE1 can be selected.
In addition, in order to prepare cells lacking an unknown hybrid sensor kinase other than those described above, for example, a hybrid sensor kinase gene of the target cell is isolated and prepared by deleting the gene by homologous recombination or the like. You can also. To isolate the hybrid sensor kinase gene of the cell of interest, the structural features of the hybrid sensor kinase can be exploited. For example, in the histidine kinase region and the receiver region, amino acid sequences around a histidine residue that can be autophosphorylated and around an aspartic acid residue that receives a phosphate group from the histidine residue are conserved. A polymerase chain reaction (hereinafter referred to as PCR) using an oligonucleotide designed based on the nucleotide sequence of the conserved region as a primer, or an oligonucleotide having a nucleotide sequence encoding the conserved amino acid sequence is used as a probe. The hybrid sensor kinase gene of the target cell can be isolated by the hybridization method. Based on the amino acid sequence deduced from the base sequence of the isolated gene, the base that encodes the amino acid sequence of the hybrid sensor kinase by verifying whether it has the above structural features It can be confirmed that the gene consists of a sequence. Specifically, the PCR method described in Srikantha, T. et.al. (1998) Microbiology 144: 2715-2729 can be mentioned. For PCR and hybridization, for example, experimental conditions used for isolating “a gene consisting of a base sequence encoding an amino acid sequence of an osmotic pressure sensitive histidine kinase that does not have a transmembrane region” described later can be used. .
Further, for example, according to the method described in Nagahashi, S. et.al. (1998) Microbiology 144: 425-432, Srikantha, T. et.al. (1998) Microbiology 144: 2715-2729, the expression of SLN1 was In a budding yeast that has been conditionally suppressed, a hybrid sensor kinase gene can be isolated using functional complementarity as an index, and cells lacking the gene can be prepared.

(Osmotic pressure sensitive histidine kinase without transmembrane region)
Next, the “osmotic pressure sensitive histidine kinase that does not have a transmembrane region” introduced in a functional manner into the “cell deficient in at least one hybrid sensor kinase” will be described.
In filamentous fungi, histidine kinase having a structure similar to the above hybrid sensor kinase has been isolated. This histidine kinase has a histidine kinase region and a receiver region that are found in hybrid sensor kinases, but is characterized by not having a transmembrane region found in many hybrid sensor kinases in the input region. Instead, it is a protein having a characteristic structure in which a polypeptide consisting of about 90 amino acids having amino acid sequence homology with each other is repeated about 6 times. The mode of signal transduction from histidine kinase has not been completely elucidated, but is known to be involved in the osmotic response.
In the present invention, “homology” refers to sequence identity between two genes or two proteins. The “homology” is determined by comparing two sequences that are optimally aligned over the region of the sequence to be compared. Here, the gene or protein to be compared may have an addition or a deletion (for example, a gap) in the optimal alignment of the two sequences. Thus, “homology” refers to, for example, FASTA [Pearson & Lipman, Proc. Natl. Acad. Sci. USA, 4, 2444-2448 (1988)], BLAST [Altschul et al., Journal of Molecular Biology, 215, 403. -410 (1990)], CLUSTAL W [Thompson, Higgins & Gibson, Nucleic Acid Research, 22, 4673-4680 (1994a)] and the like, it can be calculated by creating an alignment by performing homology analysis. The above program is, for example, the DNA Data Bank of Japan [International DNA Data Bank operated within the Center for Information Biology and DNA Data Bank of Japan (CIB / DDBJ)]. It is generally available on the homepage (http://www.ddbj.nig.ac.jp). “Homology” can also be determined using commercially available sequence analysis software. Specifically, for example, GENETYX-WIN Ver.5 (manufactured by Software Development Co., Ltd.) is used, and the homology is obtained by the Lipman-Pearson method [Lipman, DJ and Pearson, WR, Science, 227, 1435-1441, (1985)] It can be calculated by creating an alignment by performing sex analysis.
Here, as `` a structure in which a polypeptide consisting of about 90 amino acids having amino acid sequence homology with each other is present about 6 times '', for example, Alex, LA et.al. (1996) Proc. Natl. Acad. Sci. USA 93: 3416-3421, Ochiai, N. et.al. (2001) Pest Manag. Sci. 57: 437-442, Oshima, M. et.al. (2002) Phytopathology 92: 75-80 Such a structure is present on the N-terminal side of the histidine kinase region. “Structure in which an amino acid sequence consisting of about 90 amino acids is repeated about 6 times” includes, for example, the sixth repetition of an truncated amino acid sequence consisting of about 90 amino acids followed by truncation Also included is a structure in which the sequence continues, a structure in which an amino acid sequence consisting of about 90 amino acids is repeated six times, followed by a truncated seventh repeat sequence. Specifically, in the amino acid sequence of histidine kinase described in the present invention, the “structure in which an amino acid sequence consisting of about 90 amino acids having amino acid sequence homology with each other is repeated about 6 times” is the amino acid represented by SEQ ID NO: 1. The region from the 190th amino acid to the 707th amino acid in the sequence, the region from the 189th amino acid to the 706th amino acid in the amino acid sequence represented by SEQ ID NO: 16, the 176th amino acid in the amino acid sequence represented by SEQ ID NO: 41 To the 693rd amino acid, from the 192nd amino acid to the 709th amino acid in the amino acid sequence represented by SEQ ID NO: 55, from the 299th amino acid to the 911th amino acid in the amino acid sequence represented by SEQ ID NO: 68 And the like. `` Osmotically sensitive histidine kinase that does not have a transmembrane region '' is a histidine kinase that is characteristically found in filamentous fungi, and a repeated sequence region of polypeptides consisting of about 90 amino acids having amino acid sequence homology to each other, It refers to a protein having a histidine kinase region and a receiver region, having structural features that do not have a transmembrane region, and having osmotic pressure sensitivity.
In order to confirm that the protein has osmotic pressure sensitivity, it may be confirmed that the sensitivity of the cell to osmotic stress is enhanced by deleting the protein (histidine kinase) from the cell. (Histidine kinase) was introduced into the budding yeast lacking the osmotic pressure sensitive hybrid sensor kinase SLN1, and it was confirmed that the function of the deficient SLN1 was complemented and the yeast could grow, and the protein penetrated. It can also be confirmed that it is pressure sensitive.
Among filamentous fungi, there is mainly the presence of “osmotic pressure-sensitive histidine kinase that does not have a transmembrane region” in red spider mold, which is a model organism of filamentous fungi, and phytopathogenic fungi, which are pathogenic microorganisms that use plants as hosts. It has been reported.
Examples of the histidine kinase of the present invention or a gene thereof include histidine kinase having one of the following amino acid sequences or a gene thereof.
<Amino acid sequence>
(A) the amino acid sequence represented by SEQ ID NO: 41, SEQ ID NO: 55 or SEQ ID NO: 68,
(B) An osmotic pressure that is an amino acid sequence in which one or more amino acids are deleted, added, or substituted in the amino acid sequence represented by SEQ ID NO: 41, SEQ ID NO: 55, or SEQ ID NO: 68, and has no transmembrane region. The amino acid sequence of the sensitive histidine kinase,
(C) the amino acid sequence of an osmotic pressure sensitive histidine kinase which is an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown by SEQ ID NO: 41, SEQ ID NO: 55 or SEQ ID NO: 68 and which does not have a transmembrane region

  Here, “amino acid deletion, addition or substitution” in (b) and “95% or more sequence identity” in (c) include, for example, SEQ ID NO: 41, SEQ ID NO: 55, or SEQ ID NO: 68, a naturally occurring mutation caused by differences in individual processing of the organism from which the protein is derived, or an artificial amino acid mutation (for example, site-specific mutagenesis) Amino acid mutations present in the amino acid sequences of proteins produced by introducing and expressing mutations in DNA encoding natural proteins by methods, mutation treatments, and the like.

  Examples of methods for artificially performing the “amino acid deletion, addition, or substitution” in the above (b) (hereinafter sometimes collectively referred to as amino acid modification) include, for example, SEQ ID NO: 41 and SEQ ID NO: 55. Alternatively, a conventional site-directed mutagenesis is performed on the DNA encoding the amino acid sequence represented by SEQ ID NO: 68, and then this DNA is expressed by a conventional method. Here, as a site-specific mutagenesis method, for example, a method using amber mutation (gapped duplex method, Nucleic Acids Res., 12, 9441-9456 (1984)), a PCR method using a mutagenesis primer Etc.

  The number of amino acids modified as described above is at least 1 residue, specifically 1 or several (here, “several” is about 2 to about 10) or more. . The number of such modifications may be in a range where the effects of the present invention can be found.

  Of the deletions, additions or substitutions, the modification relating to amino acid substitution is particularly preferable. The substitution is more preferably substitution with an amino acid having similar properties such as hydrophobicity, charge, pK, and structural features. Examples of such substitution include (1) glycine, alanine; (2) valine, isoleucine, leucine; (3) aspartic acid, glutamic acid, asparagine, glutamine, (4) serine, threonine; (5) lysine, arginine. And (6) substitution within the group of phenylalanine and tyrosine.

  In the present invention, “sequence identity” refers to sequence identity and homology between two genes or two proteins. The “sequence identity” is determined by comparing two sequences that are optimally aligned over the entire region of the sequence to be compared. Here, the gene or protein to be compared may have an addition or a deletion (for example, a gap) in the optimal alignment of the two sequences. Such “sequence identity” is exemplified by FASTA [Pearson & Lipman, Proc. Natl. Acad. Sci. USA, 4, 2444-2448 (1988)], BLAST [Altschul et al., Journal of Molecular Biology, 215, 403-410 (1990)], CLUSTAL W [Thompson, Higgins & Gibson, Nucleic Acid Research, 22, 4673-4680 (1994a)], etc., and can be calculated by creating an alignment by performing homology analysis. . The above program is, for example, the DNA Data Bank of Japan [International DNA Data Bank operated within the Center for Information Biology and DNA Data Bank of Japan (CIB / DDBJ)]. It is generally available on the homepage (http://www.ddbj.nig.ac.jp). “Homology” can also be determined using commercially available sequence analysis software. Specifically, for example, GENETYX-WIN Ver.5 (manufactured by Software Development Co., Ltd.) is used, and the homology is obtained by the Lipman-Pearson method [Lipman, DJ and Pearson, WR, Science, 227, 1435-1441, (1985)] It can be calculated by creating an alignment by performing sex analysis.

  For example, the sequence identity in the present invention is preferably 95% or more in the case of the amino acid sequence standard, and preferably 95% or more in the case of the base sequence standard.

Hereinafter, a more specific example of “osmotic pressure sensitive histidine kinase having no transmembrane region” will be described.

(Osmotically sensitive histidine kinase that does not have a transmembrane region of apacan mold)
The protein OS-1 encoded by the OS-1 gene isolated from the osmotic pressure-sensitive mutant os-1 of red-knot mold can be cited as "osmotic-sensitive histidine kinase that does not have a transmembrane region" (Schumacher, MM et.al. (1997) Current Microbiol. 34: 340-347, Alex, LA et.al. (1996) Proc. Natl. Acad. Sci. USA 93: 3416-3421). The amino acid sequence of OS-1 and the base sequence of the OS-1 gene have been published (amino acid sequences: AAB03698, AAB01979, base sequences: U50263, U53189), and US5,939,306 is useful for screening antibacterial active substances. It is described in. Since the red-spotted mold mutant os-1 is more sensitive to hyperosmotic stress than the wild-type strain, it is known that OS-1 is an osmotic-sensitive histidine kinase involved in osmotic adaptation in red-spotted mold. OS-1 is known from the amino acid sequence to have the structural features described above. It is also known that the red mold fungus mutant os-1 has resistance to a bactericidal agent containing a dicarboximide antibacterial active substance, an aromatic hydrocarbon antibacterial active substance or a phenylpyrrole antibacterial active substance as an active ingredient. It has been. In addition, the OS-1 mutant gene isolated from the Akapan mold mutant resistant to fungicides containing dicarboximide-based antibacterial active substances as an active ingredient has an amino acid substitution in the characteristic repetitive sequence of OS-1. A gene mutation that resulted in a mutation was found on the OS-1 gene (Miller, TK et.al. (2002) Fungl Gen. Biol. 35: 147-155). For these reasons, it is expected that the antibacterial active substance contained as an active ingredient of the above bactericidal agent targets red-knot mold OS-1.

(Osmotically sensitive histidine kinase that does not have a transmembrane domain of gray mold fungi)
Next, a specific example of “osmotic pressure sensitive histidine kinase that does not have a transmembrane region” is BcOS-1 of Botryotinia fuckeliana. The BcOS-1 gene has been isolated as a homologous gene of the red-knot mold OS-1 gene, and its nucleotide sequence and amino acid sequence have been published (base sequence: Genebank accession AF396287, AF435964, amino acid sequence: Genebank accession AAL37947, AAL30826). BcOS-1 is known from the amino acid sequence to have the structural features described above. In addition, the BacOS-1 gene isolated from a strain resistant to fungicides containing dicarboximide antibacterial active substance of gray mold fungus as an active ingredient is effective against red carp mold dicarboximide antibacterial active substance As in the case of the OS-1 gene isolated from a strain resistant to the fungicide contained as a component, a genetic mutation that results in an amino acid substitution in the characteristic repeat sequence of BcOS-1 Recognized above. Furthermore, this antibacterial active substance resistant mutant lacking BcOS-1 is known to be an osmotic sensitive histidine kinase because of its higher osmotic sensitivity than the wild type (Oshima, M. et.al. (2002) Phytopathology 92: 75-80).
More specifically, examples of BcOS-1 include BcOS-1 having the amino acid sequence represented by SEQ ID NO: 1 isolated from the Bc-16 strain described in the Examples.

(Osmotic pressure sensitive histidine kinase without the transmembrane domain of rice blast fungus)
Furthermore, as a specific example of “osmotic pressure-sensitive histidine kinase having no transmembrane region”, HIK1 of rice blast fungus (Magnaporthe grisea) can be mentioned. The nucleotide sequence and amino acid sequence of the HIK1 gene are disclosed as homologous genes of the red-knot mold OS-1 gene (base sequence: Genebank accession AB041647, amino acid sequence: Genebank accession BAB40947). HIK1 is known from its amino acid sequence to have structural features such as not having a transmembrane region as described above. In addition, rice blast fungi lacking the HIK1 gene showed higher osmotic sensitivity than the wild type, indicating that HIK1 is an osmotically sensitive histidine kinase. (hppt: //www.sci.saitama-u.ac.jp/seitai/iden/Japanese/Abst Symp3.html)
More specifically, HIK1 includes HIK1 having the amino acid sequence represented by SEQ ID NO: 16 isolated from the P-37 strain described in the Examples.

(Definition of filamentous fungi and yeast)
In the present invention, “filamentous fungi” are described in “Revised Classification and Identification of Microorganisms (above), Takeharu Hasegawa, Society Publishing Center, 1984 (ISBN 4-7622-7399-6)” and the like. , Among fungi consisting of Myxomycota and Eumycota, fungi other than fungi that can be classified as yeast. For example, examples of the filamentous fungi classified as a genus Mycobacterium include Plasmodiophora brassicae, which belongs to the class of Mycophyceae. In addition, the fungi classified as Eubacteria are potato Phytophthora infestans belonging to the flagellum subfamily, Rhizopus stolonifer, and the blight fungus (Rhizopus stolonifer) belonging to the zygomycete subfamily. Rhizopus oryzae), Neurospora crassa belonging to Ascomycotina, Mycospharella tritici, wheat powdery mildew (Erysiphe graminis), rice fungus (Linocarpon cariceti) , Sesame leaf blight fungus (Cochliobolus miyabeanus), gray mold fungus fungus (Botrytinia fuckeliana), rice blast fungus fungus (Magnaporthe grisea), basidiomycetous fungus (Ustilago maydis), wheat rust Fungus fungi (Puccinia recondita), rice mold blight fungus (Thanatephorus cucumeris), tomato leaf mold fungus (Cladosporium fulvum) belonging to incomplete fungus, pear black spot fungus fungus (Alte) rnaria kikuchiana), spinach wilt fungus (Fusarium oxysporum) and the like.
In addition, yeast (yeast), as described in "Revised Classification and Identification of Microorganisms (above), Takeharu Hasegawa, Society Publishing Center, 1984 (ISBN 4-7622-7399-6)" It means fungi such as “proliferated mainly by budding, long single-cell generation, and colonies formed by single-cell growth do not become hairy, but become white or light paste”. Examples include Saccharomyces cerevisiae belonging to the genus Saccharomyces, Schizosaccharomyces pombe belonging to the genus Schizosaccharomyces, Phichia burtonii belonging to the genus Phichia, Candida albicans belonging to the genus Candida, and the like.

(It is a histidine kinase having a resistance mutation to any of a dicarboximide antibacterial substance, an aromatic hydrocarbon antibacterial substance or a phenylpyrrole antibacterial active substance, and has an osmotic pressure sensitivity without a transmembrane region. Histidine kinase)
The above-mentioned “osmotic pressure sensitive histidine kinase having no transmembrane region” has a resistance mutation to any of a dicarboximide antibacterial active substance, an aromatic hydrocarbon antibacterial active substance, or a phenylpyrrole antibacterial active substance. .
Here, the dicarboximide antimicrobial active, a generic name for an antimicrobial active substance a basic skeleton dicarboximides, Modern Selective Fungicide Properties, Applications, Mechanisms of Action- 2 nd rivesd and enlarged edition Lyr, H. ed. Gustav Fischer Verlag, New York, USA ISBN 3-334-60455-1 Capter 6, p99-118 and the like are shown. Specifically, a compound having a structure represented by the chemical formula (1) (Procymidone: hereinafter sometimes referred to as the compound (1)), a compound having a structure represented by the chemical formula (2) (Iprodione: hereinafter referred to as the compound) (2)), and a compound having a structure represented by the chemical formula (3) (Vinclozolin: hereinafter sometimes referred to as compound (3)). The aromatic hydrocarbon-based antimicrobial active, a generic name for an antimicrobial active substance a benzene ring as a basic skeleton, Modern Selective Fungicide Properties, Applications, Mechanisms of Action- 2 nd rivesd and enlarged edition Lyr, H. Ed. Gustav An antibacterial active substance described in Fischer Verlag, New York, USA ISBN 3-334-60455-1 Capter 5, p75-98, etc. Specifically, a compound having a structure represented by the chemical formula (4) (Quintozene: hereinafter also referred to as the compound (4)), a compound having a structure represented by the chemical formula (5) (Tolclofos-methyl: hereinafter And may be referred to as the compound (5)). Further, the phenyl pyrrole antimicrobial active, a generic name for an antimicrobial active substance a basic skeleton phenyl pyrrole, Modern Selective Fungicide Properties, Applications, Mechanisms of Action- 2 nd rivesd and enlarged edition Lyr, H. Ed. Gustav An antibacterial active substance described in Fischer Verlag, New York, USA ISBN 3-334-60455-1 Capter 19, p405-407 and the like. Specifically, a compound having a structure represented by chemical formula (6) (Fludioxonil: hereinafter sometimes referred to as compound (6)), a compound having a structure represented by chemical formula (7) (Fenpiclonil: hereinafter referred to as compound) (It may be described as (7)).

The chemical formulas of typical compounds of the dicarboximide antibacterial active substance, aromatic hydrocarbon antibacterial active substance and phenylpyrrole antibacterial active substance are shown below.

(1) Compound having the structure represented by chemical formula (1) (compound (1))

（２）化学式（２）に示される構造を有する化合物（化合物（２））

(2) Compound having the structure represented by chemical formula (2) (compound (2))

（３）化学式（３）に示される構造を有する化合物（化合物（３））

(3) Compound having the structure represented by chemical formula (3) (compound (3))

（４）化学式（４）に示される構造を有する化合物（化合物（４））

(4) Compound having the structure represented by chemical formula (4) (compound (4))

（５）化学式（５）に示される構造を有する化合物（化合物（５））

(5) Compound having the structure represented by chemical formula (5) (compound (5))

（６）化学式（６）に示される構造を有する化合物（化合物（６））

(6) Compound having the structure represented by chemical formula (6) (compound (6))

（７）化学式（７）に示される構造を有する化合物（化合物（７））

(7) Compound having the structure represented by chemical formula (7) (compound (7))

"Resistance mutation to any of dicarboximide antibacterial active substance, aromatic hydrocarbon antibacterial active substance or phenylpyrrole antibacterial active substance" means dicarboximide antibacterial active substance, aromatic hydrocarbon antibacterial activity A mutation that can be found in an `` osmotic pressure sensitive histidine kinase that does not have a transmembrane region '' produced by a mutant of a filamentous fungus having resistance to either a substance or a phenylpyrrole antibacterial active substance, An amino acid substitution, addition or deletion mutation involved in resistance to an imide antibacterial active substance, an aromatic hydrocarbon antibacterial active substance or a phenylpyrrole antibacterial active substance. However, mutations that cause the “osmotic pressure sensitive histidine kinase not having a transmembrane region” to function as histidine kinase due to the mutation are excluded. Here, mutants of filamentous fungi having resistance to any of dicarboximide antibacterial active substance, aromatic hydrocarbon antibacterial active substance or phenylpyrrole antibacterial active substance are dicarboximide antibacterial active substance, aromatic Even if it is a filamentous fungus that can be isolated from the natural world to which an aromatic hydrocarbon-based antibacterial active substance or phenylpyrrole-based antibacterial active substance is applied, the filamentous fungus is a dicarboximide-based antibacterial active substance, Filamentous fungi that have acquired resistance selected by, for example, artificial culture in the presence of a phenylpyrrole-based antibacterial active substance may be used.
Specifically, in BcOS-1 which is `` osmotic pressure sensitive histidine kinase that does not have a transmembrane region '' of gray mold fungus, amino acid substitution I365S involved in resistance to dicarboximide antibacterial active substance is Oshima, M. et.al. (2002) Phytopathology 92: 75-80. Here, “I365S” means that the 365th isoleucine is substituted with serine. Hereinafter, amino acid substitutions are described in the same manner. As an amino acid substitution involved in resistance to a dicarboximide antibacterial active substance in the OS-1 gene, which is an osmotic pressure sensitive histidine kinase having no transmembrane region, G580R, I582M, M639V, A578V, G580G, and L625P have been reported in Miller, TK et.al. (2002) Fungal Gen. Biol. 35: 147-155 as an amino acid deletion. Here, 680K means that the 680th lysine is deleted. Hereinafter, amino acid deletions are described in the same manner. In addition, A578V, G580R, and L625P are Ochiai, N. et.al. (2001) Pest Managenment Sci. 57: 437 as amino acid substitutions involved in resistance to phenylpyrrole antibacterial active substances in the OS-1 gene of red bread mold. Reported at -442.
More specifically, BcOS-1 having the amino acid sequence represented by SEQ ID NO: 13 described in Examples can be mentioned.
In addition to the above-mentioned resistance mutations, isolated from mutants of filamentous fungi having resistance to either dicarboximide antibacterial substances, aromatic hydrocarbon antibacterial substances or phenylpyrrole antibacterial substances A resistance mutation may be found by analyzing the amino acid sequence of “osmotic pressure sensitive histidine kinase that does not have a transmembrane region” and comparing it with the amino acid sequence of the protein in a sensitive wild type strain.

(Transformation in which a gene comprising a base sequence encoding the amino acid sequence of osmotic pressure sensitive histidine kinase not having a transmembrane region is introduced into a cell deficient in at least one hybrid sensor kinase in a functional form. Cell production)
A transformed cell into which a gene comprising a base sequence encoding the amino acid sequence of an osmotic pressure sensitive histidine kinase (hereinafter also referred to as histidine kinase of the present invention) having no transmembrane region is introduced in a functional form. Is introduced by introducing "a gene comprising a nucleotide sequence encoding the amino acid sequence of the histidine kinase of the present invention" or the like into a "cell deficient in at least one hybrid sensor kinase" as a host cell as follows. Obtainable. Hereinafter, an example of the method for producing the transformed cell will be described.

(1) Preparation of cDNA First, from filamentous fungi, for example, according to the method described in Molecular Cloning 2nd edition by J., Sambrook, E., F., Frisch, T., Maniatis. Prepare total RNA. Specifically, for example, red spruce mold (Neurospora crassa), gray mold fungus (Botrytinia fuckeliana), rice blast fungus (Magnaporthe grisea), potato blight fungus (Phytophthora infestans), rice mold blight fungus (Thanatephorus) cucumeris), spinar wilt fungus (Fusarium oxysporum), wheat leaf blight fungus (Mycospharella tritici), tomato plague fungus (Phytophthora infestans), etc. (A) A method of obtaining total RNA by adding a solution containing guanidine hydrochloride and phenol or a solution containing SDS and phenol to the obtained ground product, (b) A method of obtaining total RNA by adding a solution containing guanidine thiocyanate to the above-mentioned ground product, further adding CsCl and centrifuging may be used. For this operation, for example, a commercially available kit such as RNeasy Plant Mini Kit (manufactured by QIAGEN) can also be used.

Next, cDNA is prepared using the total RNA thus prepared. For example, after annealing an oligo dT chain or a random primer to total RNA, cDNA may be prepared by acting reverse transcriptase. Furthermore, a double-stranded cDNA can be prepared by allowing RNaseH or DNA Polymerase I to act on the cDNA. For this operation, commercially available kits such as SMART ™ PCR cDNA Synthesis Kit (Clontech), cDNA Synthesis Kit (Takara Shuzo), cDNA Synthesis Kit (Amersham Pharmacia) and ZAP-cDNA Synthesis Kit (Stratagene) are used. Can be used.

(2) In the case of a gene whose cloning base sequence is already known, for example, PCR using a oligonucleotide having a partial base sequence of the public base sequence as a primer from the cDNA prepared as described above, A gene comprising a base sequence encoding the amino acid sequence of the histidine kinase of the present invention can be obtained by a hybridization method using an oligonucleotide having a partial base sequence of the base sequence as a probe.
Specifically, PCR using an oligonucleotide having a partial base sequence of the base sequence represented by SEQ ID NO: 2 as a primer from gray mold fungus cDNA, or an oligo having a partial base sequence of the base sequence represented by SEQ ID NO: 2 A gene comprising a base sequence encoding the amino acid sequence of BcOS-1, which is the histidine kinase of the present invention, can be obtained by a hybridization method using nucleotides as probes.
Specifically, PCR using a rice blast fungus cDNA having an oligonucleotide having a partial base sequence of the base sequence shown by SEQ ID NO: 17 as a primer, or a partial base sequence of the base sequence shown by SEQ ID NO: 17 A gene comprising a base sequence encoding the amino acid sequence of HIK1, which is the histidine kinase of the present invention, can be obtained by a hybridization method using the oligonucleotide possessed as a probe.
In the case of a gene whose base sequence is unknown, a hybridization method using an oligonucleotide having a partial base sequence of the base sequence of the histidine kinase of the present invention whose base sequence is known as a probe, or a base sequence and / or an amino acid sequence A plurality of known histidine kinases of the present invention can be obtained by PCR using oligonucleotides designed based on highly homologous base sequences and / or amino acid sequences as primers. A plurality of base sequences and / or amino acid sequences having high homology among the plurality of histidine kinases of the present invention whose base sequences and / or amino acid sequences are known include, for example, “from about 90 amino acids which are structural features of the histidine kinase of the present invention. The nucleotide sequence and / or amino acid sequence of the conserved motif found in the “repeat sequence region of polypeptide”, “histidine kinase region”, “receiver region” and the like.

More specifically, when BcOS-1 of gray mold fungus is obtained by PCR, a base sequence of about 20 bp to about 40 bp, for example, a 5 ′ untranslated region of the base sequence represented by SEQ ID NO: 2 And oligonucleotides designed and synthesized based on the base sequences selected from the 3 ′ untranslated region, respectively, can be used as a primer set. As the primer set, for example, a set of an oligonucleotide having a base sequence represented by SEQ ID NO: 3 and an oligonucleotide having a base sequence represented by SEQ ID NO: 4 can be cited as a primer set. The PCR reaction solution to be used may be prepared by adding a commercially available DNA polymerase as described later or a reaction solution specified by the kit to 250 ng of cDNA. The PCR reaction conditions can be appropriately changed depending on the primer set to be used. For example, the sample is kept at 94 ° C. for 2 minutes, then kept at about 8 ° C. for 3 minutes, then at 94 ° C. for 30 seconds and then at 55 ° C. Incubate for 30 seconds and then heat at 72 ° C for 4 minutes as one cycle, repeat this for about 40 cycles, or hold at 94 ° C for 5 seconds and then at 72 ° C for 4 minutes for 1 cycle. A cycle in which the temperature is kept at 94 ° C. for 5 seconds and then kept at 70 ° C. for 4 minutes is defined as one cycle, and this can be repeated 20 to 40 cycles. Examples of such operations include Takara Heraculase ^™ (Takara Shuzo), DNA polymerase included in Advantage cDNA PCR Kit (Clontech), TAKARA Ex Taq (Takara Shuzo), PLATINUM ^™ PCR SUPER Mix (Lifetech Oriental) ) And KOD-Plus- (manufactured by Toyobo) can be used.
In addition, when HIK1 of rice blast fungus is obtained by PCR, for example, based on a base sequence selected from the 5 ′ untranslated region and the 3 ′ untranslated region of the base sequence represented by SEQ ID NO: 17, respectively. Designed and synthesized oligonucleotides can be used as primer sets. As the primer set, for example, a set of an oligonucleotide consisting of the base sequence shown by SEQ ID NO: 18 and an oligonucleotide consisting of the base sequence shown by SEQ ID NO: 19 can be cited as a primer set. As for the PCR reaction solution and reaction conditions used, the HIK1 gene can be obtained by performing PCR in the same manner as described above.
In addition, the base sequence of histidine kinase gene, such as spinach wilt fungus (Fusarium oxysporum), wheat leaf blight fungus (Mycospharella tritici), rice mold blight fungus (Thanatephorus cucumeris), tomato blight fungus (Phytophthora infestans), etc. In the case of a gene derived from a fungus whose unknown is unknown, first, a partial fragment of the gene may be obtained by PCR as described below. Primer sets include, for example, oligos designed and synthesized based on the amino acid sequences of conserved motifs found in the “repetitive sequence region”, “histidine kinase region”, “receiver region”, etc., which are structural features of this histidine kinase. A set of nucleotides can be used. Specific examples of the primer set include a primer set of an oligonucleotide having any one of the base sequences represented by SEQ ID NOs: 30 to 34 and an oligonucleotide having any one of the base sequences represented by SEQ ID NOs: 35 to 40. be able to. For example, in the case of spinach wilt disease fungus (Fusarium oxysporum), for example, using an oligonucleotide primer having the base sequence represented by SEQ ID NO: 33 and an oligonucleotide primer having the base sequence represented by SEQ ID NO: 38, KOD- Using Plus- (TOYOBO), after incubating at 94 ° C for 2 minutes, one cycle consists of 94 ° C for 15 seconds, then 55 ° C for 30 seconds and then 68 ° C for 5 minutes. To do. Similarly, in the case of wheat leaf blight fungus (Mycospharella tritici), for example, using an oligonucleotide primer having the base sequence represented by SEQ ID NO: 31 and an oligonucleotide primer having the base sequence represented by SEQ ID NO: 40, Use KOD-Plus- (TOYOBO) for 2 minutes at 94 ° C, then repeat for 35 cycles with 94 ° C for 15 seconds, 55 ° C for 30 seconds and 68 ° C for 3 minutes. Perform under conditions. In the case of rice mold blight fungus (Thanatephorus cucumeris), for example, using an oligonucleotide primer having a base sequence represented by SEQ ID NO: 30 and an oligonucleotide primer having a base sequence represented by SEQ ID NO: 37, Amplification is repeated 35 times using KOD-Plus- (TOYOBO), followed by incubation at 94 ° C for 2 minutes, then at 94 ° C for 15 seconds, then at 55 ° C for 30 seconds and then at 68 ° C for 1 minute. Perform under conditions. In the case of Phytophthora infestans, for example, an oligonucleotide primer having a base sequence represented by SEQ ID NO: 31 and an oligonucleotide primer having a base sequence represented by SEQ ID NO: 37 are used, and KOD- Using Plus- (TOYOBO), heat was kept at 94 ° C for 2 minutes, and then kept at 94 ° C for 15 seconds, then at 55 ° C for 30 seconds and then at 68 ° C for 1 minute, and this cycle was repeated 35 times. To do. Next, based on the base sequence of the partial gene fragment amplified by such PCR, the full-length gene can be obtained by using, for example, SMART RACE cDNA Amplification Kit (CLONTECH).
When the base sequence encoding the full length of the amino acid sequence of the histidine kinase of the present invention is clarified from the full-length gene obtained as described above, by PCR using an oligonucleotide having a partial base sequence of the base sequence as a primer, A gene having a base sequence encoding the amino acid sequence of the histidine kinase of the present invention can also be obtained.
Specifically, when the histidine kinase gene of the present invention of spinach wilt disease fungus (Fusarium oxysporum) is obtained by PCR, for example, from the 5 ′ end region and 3 ′ end region of the base sequence represented by SEQ ID NO: 42 Oligonucleotides designed and synthesized based on each selected base sequence can be used as a primer set. Examples of the primer set include a set of an oligonucleotide having a base sequence represented by SEQ ID NO: 52 and an oligonucleotide having a base sequence represented by SEQ ID NO: 53. PCR reaction solution used and reaction conditions are obtained in the same manner as above to obtain a polynucleotide having the base sequence encoding the amino acid sequence of the histidine kinase of the present invention derived from spinach wilt disease fungus (Fusarium oxysporum) can do.
When the histidine kinase gene of the present invention of wheat leaf blight fungus (Mycospharella tritici) is obtained by PCR, for example, it is selected from the 5 ′ end region and 3 ′ end region of the base sequence represented by SEQ ID NO: 56, respectively. Oligonucleotides designed and synthesized based on the prepared base sequence can be used as a primer set. Examples of the primer set include a set of an oligonucleotide having a base sequence represented by SEQ ID NO: 64 and an oligonucleotide having a base sequence represented by SEQ ID NO: 65. The PCR reaction solution used and the reaction conditions are as follows. By carrying out PCR in the same manner as described above, a polynucleotide having a nucleotide sequence encoding the amino acid sequence of the histidine kinase of the present invention derived from Mycospharella tritici is used. Can be acquired.
When the histidine kinase gene of the present invention of rice blight fungus (Thanatephorus cucumeris) is obtained by PCR, for example, it is selected from the 5 ′ end region and 3 ′ end region of the base sequence represented by SEQ ID NO: 69, respectively. Oligonucleotides designed and synthesized based on the prepared nucleotide sequence can be used as a primer set. Examples of the primer set include a set of an oligonucleotide having a base sequence represented by SEQ ID NO: 85 and an oligonucleotide having a base sequence represented by SEQ ID NO: 86. The PCR reaction solution used and the reaction conditions are as follows. By carrying out PCR in the same manner as described above, a polynucleotide having the base sequence encoding the amino acid sequence of the histidine kinase of the present invention derived from rice blight fungus (Thanatephorus cucumeris) is used. Can be acquired.

When using the hybridization method, for example, cloning should be performed according to the method described in Molecular Cloning 2nd edition by J., Sambrook, E., F., Frisch, T., Maniatis. Can do.
BcOS-1 of a gray mold fungus or a probe used for obtaining an unknown gene of the histidine kinase of the present invention is a DNA having a partial base sequence of the base sequence represented by SEQ ID NO: 2 (about 200 bases to The DNA is synthesized with, for example, Random Primed DNA Labeling Kit (Boehringer), Random Primer DNA Labeling Kit Ver.2 (Takara Shuzo), ECL Direct Nucleic Acid Labeling and Ditection It can be obtained by radioisotope labeling or fluorescent labeling according to a known method using System (Amersham Pharmacia), Megaprime DNA-labelling system (Amersham Pharmacia) or the like. The probe thus obtained can be used, for example, for cloning the gene of the histidine kinase of the present invention whose base sequence is known, such as the BcOS-1 gene of gray mold fungi, or the gene of the histidine kinase of the present invention whose base sequence is unknown. Can be used. Examples of hybridization conditions include stringent conditions. Specifically, for example, 6 × SSC (0.9 M NaCl, 0.09 M trisodium citrate), 5 × Denhardt's solution (0.1% (w / v) Ficoll 400, 0.1% (w / v) polyvinylpyrrolidone, 0.1% BSA), 0.5% (w / v) SDS and 100 μg / ml denatured salmon sperm DNA or 100 μg / ml denatured salmon sperm DNA Incubate in DIG EASY Hyb solution (Boehringer Mannheim) at 65 ° C, then incubate for 15 minutes at room temperature in the presence of 1x SSC (0.15M NaCl, 0.015M trisodium citrate) and 0.5% SDS The condition can be raised twice and further maintained at 68 ° C. for 30 minutes in the presence of 0.1 × SSC (0.015 M NaCl, 0.0015 M trisodium citrate) and 0.5% SDS.
More specifically, for example, to obtain the gray mold fungus BcOS-1 gene, TAKARA LA taq ^TM (manufactured by Takara Shuzo Co., Ltd.) using the gray mold fungus cDNA library phage solution (about 1,000,000 pfu) as a template. ), And by performing PCR using an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 9 and an oligonucleotide consisting of the base sequence corresponding to the complementary strand of the base sequence shown in SEQ ID NO: 10 as a primer set, The DNA to be amplified may be amplified and obtained. The PCR reaction solution to be used may be prepared by adding the reaction solution specified by the kit to 250 ng of the DNA library.
As PCR reaction conditions, for example, the sample is kept at 94 ° C. for 2 minutes, then kept at 8 ° C. for 3 minutes, and then cycles of 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 68 ° C. for 5 minutes are repeated 40 cycles. Thus, conditions for amplification can be raised.
Next, using the amplified and obtained DNA as a template and using the Megaprime DNA-labelling system (manufactured by Amersham Pharmacia), create a probe labeled with ³² P by using the reaction solution specified by the kit. Can do. Colony hybridization was performed according to the usual method using the probe thus prepared, and 6 × SSC (0.9 M NaCl, 0.09 M trisodium citrate), 5 × Denhardt's solution (0.1% (w / v) Ficoll) 400, 0.1% (w / v) polyvinylpyrrolidone, 0.1% BSA), 0.5% (w / v) SDS and 100 μg / ml denatured salmon sperm DNA or DIG EASY Hyb containing 100 μg / ml denatured salmon sperm DNA Incubate in solution (Boehringer Mannheim) at 65 ° C, then incubate twice for 15 minutes at room temperature in the presence of 1x SSC (0.15M NaCl, 0.015M trisodium citrate) and 0.5% SDS, Furthermore, a clone that hybridizes to the probe can be obtained by incubation at 68 ° C. for 30 minutes in the presence of 0.1 × SSC (0.015 M NaCl, 0.0015 M sodium citrate) and 0.5% SDS.

In addition, the gene of the histidine kinase of the present invention whose base sequence is known can be obtained, for example, based on the published base sequence by, for example, the phosphite triester method (Hunkapiller, M. et al., Nature, 310, 105, 1984) and the like, and can also be prepared by chemical synthesis of nucleic acids.

The gene consisting of the base sequence encoding the amino acid sequence of the histidine kinase of the present invention obtained as described above is `` Molecular Cloning: A Laboratory Manual 2nd edition '' (1989), Cold Spring Harbor Laboratory Press, `` Current Protocols In Molecular Biology "(1987), John Wiley & Sons, Inc. ISBN0-471-50338-X, etc. Examples of the vector used include pBlueScriptII vector (Stratagene), pUC18 / 19 vector (Takara Shuzo), TA cloning vector (Invitrogen) and the like.
The base sequence of the cloned gene is determined by Maxam Gilbert method (for example, described in Maxam, AM & W. Gilbert, Proc. Natl. Acad. Sci. USA, 74, 560, 1977 etc.) or Sanger method ( For example, Sanger, F. & ARCoulson, J. Mol. Biol., 94, 441, 1975, Sanger, F, & Nicklen and ARCoulson., Proc. Natl. Acad. Sci. USA, 74, 5463, 1977 etc. Etc.). For this operation, for example, a commercially available kit such as Termo Seqenase II dye terminator cycle sequencing kit (Amersham Pharmacia), Dye Terminator Cycle Sequencing FS Ready Reaction Kit (PE Biosystems Japan) can be used.

(3) Construction of expression vector The construction of an expression vector for a gene comprising a base sequence encoding the amino acid sequence of the histidine kinase of the present invention is carried out by a conventional method (for example, J., Sambrook, E., F., Frisch, T. The method described in Maniatis, Molecular Cloning 2nd edition, published by Cold Spring Harbor Laboratory press, etc.).
For example, a vector that can be used in the host cell to be transformed, for example, a vector that contains genetic information that can be replicated in the host cell and can be propagated autonomously, and can be isolated and purified from the host cell. Yes, and a vector that may have a detectable marker (specifically, when a bacterium such as Escherichia coli is used as a host cell, for example, plasmid pUC119 (manufactured by Takara Shuzo Co., Ltd.) or phagemid pBluescriptII (Stratagene) When yeast is used as a host cell, for example, plasmid pACT2 (manufactured by Clontech), p415CYC (ATCC87382), p415ADH (ATCC87374), etc. may be used. In this case, for example, plasmid pBI221 (Clontech) or the like may be used.) A gene comprising a base sequence encoding the amino acid sequence of the histidine kinase of the present invention is incorporated. It may be constructed by.

The amino acid sequence of the histidine kinase of the present invention is incorporated into the vector in such a manner that a promoter capable of functioning in the host cell is operably linked upstream of the gene consisting of the base sequence encoding the amino acid sequence of the histidine kinase of the present invention. It is possible to construct an expression vector that allows a gene consisting of a nucleotide sequence encoding s to be expressed in a host cell. Here, “to bind in a functional manner” means that the promoter and the histidine of the present invention are expressed in a host cell so that the gene comprising the base sequence encoding the amino acid sequence of the histidine kinase of the present invention is expressed under the control of the promoter. It means to bind a gene consisting of a base sequence encoding the amino acid sequence of a kinase. Examples of promoters that can function in the host cell include, for example, when the host cell is E. coli, the E. coli lactose operon promoter (lacP), the tryptophan operon promoter (trpP), the arginine operon promoter (argP), and the galactose operon. Promoters (galP), tac promoter, T7 promoter, T3 promoter, λ phage promoter (λ-pL, λ-pR), and the like. Further, when the host cell is yeast, the ADH1 promoter and CYC1 promoter (note that the ADH1 promoter is prepared from a yeast expression vector p415ADH (ATCC87374) having, for example, the ADH1 promoter and CYC1 terminator by a conventional genetic engineering method. The CYC1 promoter can be prepared from p415CYC (ATCC87382) by a conventional genetic engineering method. When the host cell is a plant cell, examples include nopaline synthase gene (NOS) promoter, octopine synthase gene (OCT) promoter, cauliflower mosaic virus (CaMV) -derived 19S promoter, CaMV-derived 35S promoter, and the like. it can.

In addition, when a gene comprising a base sequence encoding the amino acid sequence of the histidine kinase of the present invention is incorporated into a vector having a promoter capable of functioning in the host cell in advance, the promoter possessed by the vector and the gene of the histidine kinase of the present invention And the histidine kinase gene of the present invention may be inserted downstream of the promoter. For example, the yeast plasmid p415ADH described above has an ADH1 promoter, and if the gene of the histidine kinase of the present invention is inserted downstream of the ADH1 promoter of the plasmid, the gene of the histidine kinase of the present invention, for example, Saccharomyces cerevisiae AH22 ( Expression vectors that can be expressed in budding yeast such as IFO10144) and TM182 (Maeda, T. et.al. (1994) Nature 369: 242-245) can be constructed.

(4) Preparation of transformed cell By introducing the constructed expression vector into a host cell as a usual method, the transformed cell used in the present invention can be prepared. Examples of host cells used for producing such transformed cells include bacteria, yeast, plant cells and the like. Examples of bacteria include Escherichia coli and Vibrio harveyi. Examples of yeast include budding yeast and fission yeast, and more specifically, yeasts belonging to the genus Saccharomyces, Schizosaccharomyces, and the like. Examples of plant cells include plant cells such as Arabidopsis thaliana.
As a method for introducing the expression vector into the above host cell, a normal introduction method according to the host cell to be transformed can be applied. For example, when a bacterium is used as a host cell, the usual introduction such as the calcium chloride method and the electroporation method described in “Molecular Cloning” (J. Sambrook et al., Cold Spring Harbor, 1989), etc. The expression vector can be introduced into a host cell by using the method. When yeast is used as the host cell, the expression vector can be introduced into the host cell by using, for example, a Yeast transformation kit (manufactured by Clontech) based on the lithium method. When plant cells are used as host cells, for example, an Agrobacterium infection method (JP-B-2-58917 and JP-A-60-70080), an electroporation method to protoplasts (JP-A-60-251887 and JP-A-60-251887) The expression vector can be introduced into a host cell by using a conventional introduction method such as JP-A-5-68575) and particle gun method (Japanese Patent Publication No. 5-508316 and JP-A-63-258525).

(Intracellular signal transmission system related to histidine kinase of the present invention)
In the present invention, in order to measure the amount of intracellular signal transduction from the histidine kinase of the present invention expressed in the transformed cell prepared as described above or an index value correlated therewith, a transformed cell is prepared. An intracellular signal transmission system inherent in the host cell used for this purpose may be used. Examples of intracellular signal transmission systems that can be used include intracellular signal transmission for osmotic pressure control of budding yeast, intracellular signal transmission for the cell cycle and oxidative stress response of fission yeast, and capsular polysaccharide biosynthesis of Escherichia coli. Intracellular signal transmission related to operon expression control, intracellular signal transmission related to cell density-sensitive luminescence control of bioluminescent marine microorganism Vibrio harveyi, intracellular signal transmission related to cytokinin response of Arabidopsis thaliana, and the like.

In addition, “a cell deficient in at least one or more hybrid sensor kinases” is used as a host cell used for producing such a transformed cell. That is, since the host cell lacks at least one hybrid sensor kinase specific to the host cell, the introduced histidine kinase of the present invention functions in place of the defective hybrid sensor kinase and functions in the cell. For example, when a test substance is given to the transformed cell, a change in growth amount, a change in shape, a change in shape, a change in the amount of biosynthesis of a specific substance in the cell, Changes in the metabolism of specific substances may occur. In such a case, the antibacterial activity of the test substance acting on the histidine kinase of the present invention is changed in the amount of growth of the transformed cell, change in shape, change in shape, change in the amount of biosynthesis of the specific substance in the cell, It can be measured by changing the metabolic amount of a specific substance.
On the other hand, in the host cell used for producing the transformed cell, when at least one hybrid sensor kinase specific to the host cell is not deficient, intracellular signal transduction of the transformed cell is performed in the host cell. Signal transmission from the unique hybrid sensor kinase and intracellular signal transmission from the introduced histidine kinase of the present invention coexist. In the transformed cells, changes in the growth, morphology, and shape of the transformed cells reflecting the amount of intracellular signal transduction from the introduced histidine kinase of the present invention, biosynthesis of specific substances in the cells Changes in the amount, changes in the metabolic amount of a specific substance, and the like are affected by the amount of intracellular signal transmission from the hybrid sensor kinase specific to the host cell, and become smaller. In the present invention, by using a host cell deficient in at least one hybrid sensor kinase specific to the host cell, the transformed cell reflecting the amount of intracellular signal transduction from the introduced histidine kinase of the present invention Changes in the amount of growth, changes in shape, changes in shape, changes in the amount of biosynthesis of specific substances in cells, changes in the metabolic amount of specific substances, etc. Sensitivity is enhanced. Thus transformed cells with enhanced sensitivity to antibacterial active substances are useful for testing antibacterial activity of test substances and searching for antibacterial active substances using the test.
Specifically, a strain lacking the SLN1 gene comprising a base sequence encoding the amino acid sequence of a protein having an osmotic pressure sensor function derived from budding yeast such as Saccharomyces cerevisiae (Maeda T et al. Nature: 369 242-245 ( 1994)), when the histidine kinase of the present invention is introduced, the histidine kinase of the present invention performs signal transduction instead of the deficient SLN1, whereby the amount of intracellular signal transduction from the introduced histidine kinase of the present invention is reduced. The amount of growth can be detected more clearly as an index. That is, when the test substance acts on the histidine kinase of the present invention and the signal transduction amount in the host cell from the histidine kinase of the present invention changes, it can be clearly measured as a change in the growth amount of the transformed budding yeast. In addition, a strain deficient in the RcsC gene, which is a hybrid sensor kinase derived from Escherichia coli, a strain deficient in the PHK1-3 gene involved in cell cycle control in fission yeast, a LuxN deficient strain involved in cell density-sensitive luminescence control in Vibrio harveyi, and a cytokinin in Arabidopsis thaliana Receptor CRE1-deficient strains and the like can also be mentioned as an example of a preferred embodiment of “cells deficient in at least one hybrid sensor kinase”.

(Test method for antibacterial activity of test substance)
As a specific example of the first step of culturing transformed cells into which a gene comprising a nucleotide sequence encoding the amino acid sequence of histidine kinase of the present invention has been introduced in the test substance antibacterial activity assay method in the presence of the test substance For example, a method of bringing the test substance into contact with the transformed cell by culturing the transformed cell in a medium containing the test substance can be mentioned. The transformed cells may be cultured in any form such as liquid culture in a liquid medium or solid culture in which agar or the like is added to the liquid medium. The concentration of the test substance in the solution can be, for example, about 1 nM to about 1 mM, and preferably about 10 nM to about 100 μM. The culture time can be, for example, about 1 hour or more and about 3 days, and preferably about 25 hours to 2 days. When testing the antibacterial activity of the test substance, the medium containing the test substance may be a medium not containing the antibacterial active substance.

  The amount of intracellular signal transduction from the histidine kinase expressed in the transformed cells cultured in the first step or an index value correlated therewith is measured. Then, the antibacterial activity of the test substance is evaluated based on the difference between the intracellular signal transmission amount measured in the second step or the index value correlated therewith and the control. For example, it is preferable that at least one substance having no antibacterial activity among two or more different substances (for example, two or more different substances) measured as described above is preferable. ) Can be evaluated on the basis of the difference obtained by comparing the intracellular signal transduction amount or the index value correlated therewith in the group using each of which is independently used.

Specifically, for example, the TM182 (SLN1Δ) strain (Maeda T), which is a SLN1 gene-deficient strain into which the PTP2 Tyrosine phosphatase gene (Ota et al, Proc. NAsci. USA, 89, 2355-2359 (1992)) has been introduced. et al. Nature: 369 242-245 (1994)) as a host cell (ie, a trait having a function of directly controlling cell growth by intracellular signal transduction from the histidine kinase of the present invention). When using transformed cells, measure the antibacterial activity using the growth amount of the transformed cells in a medium (agar medium or liquid medium) using glucose as a carbon source, for example, Glu-Ura-Leu medium as an index do it. When a Glu-Ura-Leu medium (medium containing no antibacterial active substance) to which a test substance is added is used, it can be evaluated that the test substance that inhibits the growth of the transformed cells has antibacterial activity. As a control, the growth of the transformed cells in a medium using galactose instead of glucose as a carbon source, for example, a Gal-Ura-Leu medium, was examined regardless of the presence or absence of the test substance. Also good.

Using transformed cells produced using fission yeast, a Phks gene-deficient strain, as a host cell (that is, a transformed cell having a function in which the cell cycle is directly controlled by intracellular signal transmission from the histidine kinase of the present invention) In this case, the division mode of the fission yeast may be observed under a microscope. When a medium containing a test substance and not containing a substance having antibacterial activity is used, it can be evaluated that a test substance that can shorten the cell length of the dividing cells of the transformed cell has antibacterial activity.
Next, when using transformed cells prepared using RcsC gene-deficient Escherichia coli introduced with cps-LacZ as a host cell, the color development of X-Gal may be observed in an agar medium or a liquid medium (Suzuki et al.). al. Plant Cell Physiol 42: 107-113 (2001)). When a medium containing a test substance and not containing a substance having antibacterial activity is used, it can be evaluated that a test substance capable of coloring the transformed cell in blue has antibacterial activity.
In addition, a transformed cell produced using LuxN gene-deficient V. harveyi as a host cell (that is, a transformed cell having a function in which bioluminescence is directly controlled by intracellular signal transmission from the histidine kinase of the present invention) is used. In this case, the fluorescence emitted from the transformed microorganism may be observed. When a medium containing a test substance and not containing a substance having antibacterial activity is used, it can be evaluated that a test substance that can cause the transformed cells to emit fluorescence has antibacterial activity.

Furthermore, an antibacterial active substance can be searched by selecting an antibacterial active substance based on the antibacterial activity evaluated by the above-described assay method.

EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated in detail, this invention is not limited to these.

Example 1 (Isolation of an osmotic-sensitive histidine kinase BcOS-1 gene having no transmembrane region of gray mold fungus)
First, total RNA was prepared from gray mold. 100 mg of mycelium of Botryotinia fuckeliana Bc-16 grown on potato dextrose agar medium (PDA medium, Nissui Pharmaceutical) was scraped with a micro spatula using a mortar and pestle in liquid nitrogen And crushed. RNA was isolated from the frozen crushed powder using RNeasy Plant Mini Kit (QIAGEN). The frozen crushed powder was transferred to a 50 ml sample tube together with liquid nitrogen. After the liquid nitrogen had evaporated, the solution with 10 μl of mercaptoethanol per ml was added to the buffer RLC attached to the kit and stirred. . Further, the pulverized powder was well dispersed by several pipettings and kept at 56 ° C. for 3 minutes. Thereafter, the solution containing the crushed powder was applied to a QIAshredder spin column attached to the kit and centrifuged at 8,000 × g for 2 minutes. The filtered supernatant was transferred to a new sample tube, 0.5 volume of 99.5% ethanol was added and mixed well by pipetting. This mixed solution was subjected to RNeasy mini spin column attached to the kit and centrifuged at 8,000 × g for 1 minute. The filtrate was discarded, 700 μl of buffer RW1 attached to the kit was added, and the mixture was centrifuged at 8,000 × g for 1 minute, and the filtrate was discarded. Furthermore, 500 μl of buffer RPE attached to the kit was added and centrifuged at 8,000 × g for 1 minute, and the filtrate was discarded. This operation was repeated twice. Finally, the upper filter portion was transferred to a new sample tube, subjected to 30 μl of RNase-free sterilized water attached to the kit, and centrifuged at 8,000 × g for 1 minute to elute the total RNA in the filtrate. This elution operation was repeated twice. The eluted total RNA had a concentration of 322 μg / ml from an absorbance at 260 nm.
Next, cDNA was synthesized using the ThermoScript RT-PCR System (Invitrogen) using total RNA as a template. A solution in which 2.7 μl of total RNA and 6.3 μl of sterilized distilled water were mixed with 1.0 μl of 50 mM Oligo (dT) ₂₀ attached to the kit and _2.0 μl of 10 mM dNTP Mix was treated at 65 ° C. for 5 minutes and rapidly cooled on ice. To this solution, add 4 μl of 5x cDNA Synthesis Buffer attached to the kit, 1 μl of 0.1M DTT, 1 μl of RNase OUT, 1 μl of ThermoScript RT, 1 μl of sterile distilled water, and react at 50 ° C. for 60 minutes. The reaction was stopped by heat treatment at 5 ° C. for 5 minutes. Further, 1 μl of RNaseH attached to the kit was added to this reaction solution, and reacted at 37 ° C. for 20 minutes to decompose the template RNA and synthesize cDNA.
Using this cDNA as a template, the BcOS-1 gene of gray mold fungus was amplified by PCR. PCR reaction was performed using the oligonucleotide consisting of the base sequence shown in SEQ ID NO: 3 and the oligonucleotide consisting of the base sequence shown in SEQ ID NO: 4 as primers to amplify the DNA having the base sequence shown in SEQ ID NO: 2. PCR reaction was carried out using KOD-Plus- (TOYOBO) at 94 ° C for 2 minutes, followed by 35 cycles of 94 ° C for 15 seconds, 55 ° C for 30 seconds, and 68 ° C for 6 minutes. Went under. The PCR reaction solution (50 μl) was 2 μl of the above cDNA, 5 μl of 10 × Buffer, 5 μl of 2 mM dNTPs, 2 μl of 25 mM MgSO ₄ , 1 μl of 10 μM oligonucleotide primer, 33 μl of sterile distilled water, KOD-Plus- Prepared by adding 1 μl. After the reaction, a part of the reaction solution was separated by 0.8% agarose gel electrophoresis, and it was confirmed that about 4 kb of BcOS-1 gene was amplified by staining with ethidium bromide.

Example 2 (Construction of expression plasmid of BcOS-1 gene of gray mold fungus and preparation of transformed yeast)
The BcOS-1 gene of gray mold fungus was cloned into the yeast-E. Coli shuttle vector p415ADH (ATCC87312). The reaction solution containing the BcOS-1 gene fragment prepared in Example 1 was purified using QIAquick PCR Purification Kit (QIAGEN) according to the attached manual. The purified BcOS-1 gene is digested with restriction enzymes SpeI and PstI. At the same time, the shuttle vector p415ADH is also digested with restriction enzymes SpeI and PstI and then separated by 0.8% agarose gel electrophoresis to contain the desired DNA. The gel part was cut out. Using QIAquick Gel Extraction Kit (QIAGEN), a gene fragment containing BcOS-1 digested with restriction enzymes SpeI and PstI and a shuttle vector digested with restriction enzymes SpeI and PstI were recovered from the gel according to the attached manual. Using the Ligation Kit Ver.2 (TaKaRa), the BcOS-1 gene fragment was inserted between SpeI and PstI of the multiple cloning site of the shuttle vector to construct the expression plasmid pADHBcOS1. The base sequence of the obtained expression plasmid was subjected to a sequencing reaction using the BigDye terminator v3.0 Cycle Sequencing Ready Reaction Kit (Applied Biosystems) according to the attached manual, and analyzed with a DNA sequencer (model 3100, Applied Biosystems). In the sequencing reaction, an oligonucleotide consisting of any one of the nucleotide sequences shown in SEQ ID NOs: 5 to 12 is used as a primer, and a heat retention cycle of 96 ° C. for 10 seconds, 50 ° C. for 5 seconds, and 60 ° C. for 4 minutes is repeated 30 times. Performed under amplification conditions. As a result, the base sequence shown in SEQ ID NO: 2 was obtained, and it was confirmed that the expression plasmid pADHBcOS1 possesses the BcOS-1 gene.
The prepared expression plasmid pADHBcOS1 was prepared according to the method described in Geitz RD & Woods RA (1994) Molecular Genetics of Yeast: Practical Approaches ed. Johnson JA, Oxford University Press p124-134. The gene was introduced into the TM182 strain described in Maeda T et.al. (1994) Nature vol.369, p242-245. In the obtained transformed budding yeast, the auxotrophy of leucine disappears, and the transformed budding yeast AH22 strain (AH22-BcOS1) is selected on the Glu-Leu agar medium, and the transformed budding yeast strain TM182 (TM182 -BcOS1) was selected on Gal-Ura-Leu agar medium. The obtained TM182-BcOS1 was confirmed to grow even when transplanted to Glu-Ura-Leu medium.

Example 3 (Antimicrobial susceptibility test of transformed budding yeast)
The transformed budding yeast AH22-BcOS1 prepared in Example 2 was cultured with shaking in Glu-Leu medium at 30 ° C. for 18 hours. As a control, the AH22 strain was similarly cultured with shaking in Glu medium at 30 ° C. for 18 hours. The absorbance at 600 nm in each of the grown transformed budding yeast cell suspensions was measured, and a cell suspension diluted with sterilized distilled water was prepared so that the absorbance was 0.1. Furthermore, a bacterial suspension in which transformed budding yeast AH22-BcOS1 was diluted 200 times with Glu-Leu medium and a bacterial suspension in which AH22 strain was diluted 200 times with Glu medium were prepared. A solution in which three types of compounds represented by chemical formulas (1) to (3) (compounds (1) to (3)) are dissolved in dimethyl sulfoxide (DMSO) so as to have a concentration of 60 ppm each, ) And (5) two compounds (compounds (4) and (5)) dissolved in dimethyl sulfoxide (DMSO) to a concentration of 2000 ppm, and chemical formulas (6) and (6) Prepare a solution in which the two compounds shown in (7) (compounds (6) and (7)) are dissolved in dimethyl sulfoxide (DMSO) to a concentration of 20 ppm. As a result, two microplates were prepared in which DMSO and 2.0 μl were dispensed into each hole. On one of them, 200 μl each of the suspension of transformed budding yeast AH22-BcOS1 diluted as described above was dispensed and left to stand at 30 ° C. for 48 hours. On the other sheet, 200 μl of the cell suspension of the control yeast AH22 strain diluted as described above was dispensed and incubated at 30 ° C. for 48 hours. After incubation, the absorbance at 600 nm in each hole was measured with a microplate reader.
Similarly, the transformed budding yeast TM182-BcOS1 prepared in Example 2 was cultured in Glu-Ura-Leu medium at 30 ° C. for 18 hours. The absorbance at 600 nm of the bacterial suspension of the grown transformed budding yeast was measured, and a bacterial suspension diluted with sterile distilled water was prepared so that the absorbance was 0.1. Furthermore, a bacterial suspension in which transformed budding yeast TM182-BcOS1 was diluted 200-fold with Glu-Ura-Leu medium and a bacterial suspension diluted 200-fold with Gal-Ura-Leu medium as a control. Prepared. A solution in which three types of compounds represented by chemical formulas (1) to (3) (compounds (1) to (3)) are dissolved in dimethyl sulfoxide (DMSO) so as to have a concentration of 60 ppm each, ) And (5) two compounds (compounds (4) and (5)) dissolved in dimethyl sulfoxide (DMSO) to a concentration of 2000 ppm, and chemical formulas (6) and (6) Prepare a solution in which the two compounds shown in (7) (compounds (6) and (7)) are dissolved in dimethyl sulfoxide (DMSO) to a concentration of 20 ppm. As DMSO, two microplates were prepared by dispensing 2.0 μl of each in two holes (that is, 2 wells). On one of them, 200 μl each of the suspension of transformed Saccharomyces cerevisiae TM182-BcOS1 diluted in Glu-Ura-Leu medium as described above was dispensed and cultured at 30 ° C. for 67 hours. . On the other side, as described above, 200 μl each of the suspension of transformed budding yeast TM182-BcOS1 diluted in Gal-Ura-Leu medium as a control was dispensed and statically cultured at 30 ° C. for 67 hours. did. After incubation, the absorbance at 600 nm in each hole was measured with a microplate reader.
Table 1 shows the degree of growth of the transformed budding yeast and the control budding yeast for the compounds represented by chemical formulas (1) to (7) (compounds (1) to (7)). The degree of growth of both the transformed budding yeast and the control budding yeast were expressed as percentages, with the absorbance at 600 nm when the compound concentration was 0 ppm being 100. TM182-BcOS1 growth inhibition by each test substance is greater than AH22-BcOS1 growth inhibition by TM182-BcOS1 It was confirmed to be a cell.

Example 4 (Isolation of BcOS-1 mutant gene of gray mold fungus resistant to dicarboximide antibacterial active substance)
Using the cDNA prepared in Example 1 as a template, BcOS of a gray mold fungus having resistance to a dicarboximide antibacterial active substance described in Oshima, M. et.al. (2002) Phytopathology 92, p75-80 A -1 mutant gene (hereinafter sometimes referred to as a BcOS-1 mutant gene) was prepared by PCR. The first PCR reaction was carried out using the oligonucleotide consisting of the base sequence shown in SEQ ID NO: 15 and the oligonucleotide consisting of the base sequence shown in SEQ ID NO: 4 as primers, and base numbers 1081 to 1081 of the base sequence shown in SEQ ID NO: 14 A DNA having the base sequence represented by 3948 was amplified. PCR reaction was carried out using KOD-Plus- (TOYOBO) at 94 ° C for 2 minutes, followed by 35 cycles of 94 ° C for 15 seconds, 55 ° C for 30 seconds, and 68 ° C for 6 minutes. Went under. The PCR reaction solution (50 μl) was 2 μl of the above cDNA, 5 μl of 10 × Buffer, 5 μl of 2 mM dNTPs, 2 μl of 25 mM MgSO ₄ , 1 μl of 10 μM oligonucleotide primer, 33 μl of sterile distilled water, KOD-Plus- Prepared by adding 1 μl. After the reaction, the second PCR was carried out using the cDNA prepared in Example 1 as a template and an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 3 and 1 μl of the reaction solution as primers. The reaction conditions were the same as in the first PCR. After the reaction, a portion of the reaction solution was separated by 0.8% agarose gel electrophoresis and stained with ethidium bromide to amplify an approximately 4 kb BcOS-1 mutant gene. I confirmed.

Example 5 (Construction of Expression Plasmid of Gray Mold Fungus BcOS-1 Mutant Gene Resistant to Dicarboximide Antibacterial Active Substance and Production of Transformed Budding Yeast)
First, the gray mold fungus BcOS-1 mutant gene (ie, BcOS-1 mutant gene) showing resistance to a dicarboximide antibacterial active substance was subcloned into the vector pBluescript II SK (+) (TOYOBO). The reaction solution containing the BcOS-1 mutant gene prepared in Example 4 was purified using QIAquick PCR Purification Kit (QIAGEN) according to the attached manual. The purified BcOS-1 mutant gene of about 4 kb was digested with restriction enzymes SpeI and PstI, while the vector pBluescript II SK (+) was also digested with restriction enzymes SpeI and PstI and then separated by 0.8% agarose gel electrophoresis. Then, the gel part containing the target DNA was cut out. Using the QIAquick Gel Extraction Kit (QIAGEN) according to the attached manual, the gene fragment containing the BcOS-1 mutant gene digested with the restriction enzymes SpeI and PstI and the vector pBluescript II SK digested with the restriction enzymes SpeI and PstI from the gel (+) Was recovered. Using the Ligation Kit Ver.2 (TaKaRa), according to the attached manual, the BcOS-1 mutant gene is inserted between SpeI and PstI of the multicloning site of the vector pBluescript II SK (+), and the plasmid pBcOS1- I365S was built. The nucleotide sequence of the obtained plasmid was subjected to a sequence reaction using BigDye terminator v3.0 Cycle Sequence FS Ready Reaction Kit (Applied Biosystems) according to the attached manual, and analyzed with a DNA sequencer (model 3100, Applied Biosystems). The sequencing reaction uses an oligonucleotide consisting of any one of the nucleotide sequences shown in SEQ ID NOs: 7 to 12 as a primer, and repeats 30 cycles of 96 ° C. for 10 seconds, 50 ° C. for 5 seconds, and 60 ° C. for 4 minutes. Performed under conditions. As a result, the base sequence shown in SEQ ID NO: 14 was obtained, and it was confirmed that the plasmid pBcOS1-I365S carries the BcOS-1 mutant gene.
The BcOS-1 mutant gene contained in the plasmid pBcOS1-I365S thus prepared was cloned into the yeast-E. Coli shuttle vector p415ADH to construct an expression plasmid. Plasmid pBcOS1-I365S was digested with restriction enzymes SpeI and PstI, while shuttle vector p415ADH was also digested with restriction enzymes SpeI and PstI. These were each separated by 0.8% agarose gel electrophoresis, and then the gel part containing the BcOS-1 mutant gene fragment digested with restriction enzymes SpeI and PstI and the shuttle vector p415ADH digested with restriction enzymes SpeI and PstI was cut out, and QIAquick Using the Gel Extraction Kit (QIAGEN), the BcOS-1 mutant gene fragment digested with the restriction enzymes SpeI and PstI and the shuttle vector digested with the restriction enzymes SpeI and PstI were collected from the gel according to the attached manual. Using Ligation Kit Ver.2 (TaKaRa), according to the attached manual, the BcOS-1 mutant gene was inserted between SpeI and PstI of the multicloning site of the shuttle vector to construct an expression plasmid pADHBcOS1-I365S. The nucleotide sequence of the obtained expression plasmid was analyzed with a DNA sequencer (model 3100, Applied Biosystems) using BigDye terminator v3.0 Cycle Sequence FS Ready Reaction Kit (Applied Biosystems) according to the attached manual. The sequencing reaction uses an oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NOs: 5 to 12 as a primer, and amplification conditions for 30 cycles of 96 ° C. for 10 seconds, 50 ° C. for 5 seconds, and 60 ° C. for 4 minutes. I went there. As a result, the base sequence shown in SEQ ID NO: 14 was obtained, and it was confirmed that the expression plasmid pADHBcOS1-I365S carries the BcOS-1 mutant gene.
The prepared expression plasmid pADHBcOS1-I365S was introduced into Saccharomyces cerevisiae TM182 according to the method described in Example 2. In the resulting transformed budding yeast, the transformed budding yeast strain TM182 (TM182-BcOS1-I365S) was selected on a Gal-Ura-Leu agar medium, taking advantage of the loss of leucine auxotrophy. The obtained TM182-BcOS1-I365S was confirmed to grow even when transplanted to Glu-Ura-Leu medium.

Example 6 (Antimicrobial susceptibility test of transformed budding yeast TM182-BcOS1-I365S)
The transformed budding yeast TM182-BcOS1-I365S prepared in Example 5 was cultured in Glu-Ura-Leu medium at 30 ° C. for 18 hours. The absorbance at 600 nm of the bacterial suspension of the grown transformed budding yeast is measured, and a bacterial suspension diluted with sterile distilled water is prepared so that the absorbance is 0.1. Furthermore, a bacterial suspension in which transformed budding yeast TM182-BcOS1-I365S was diluted 200-fold with Glu-Ura-Leu medium and a bacterial suspension diluted 200-fold with Gal-Ura-Leu medium as a control And prepare. A solution in which three types of compounds represented by chemical formulas (1) to (3) (compounds (1) to (3)) are dissolved in dimethyl sulfoxide (DMSO) to a concentration of 60 ppm each, ) And (5) two compounds (compounds (4) and (5)) dissolved in dimethyl sulfoxide (DMSO) to a concentration of 2000 ppm, and chemical formulas (6) and Prepare a solution in which the two compounds shown in (7) (compounds (6) and (7)) are dissolved in dimethyl sulfoxide (DMSO) to a concentration of 20 ppm. As a result, two microplates were prepared in which DMSO and 2.0 μl were dispensed into each hole. On one of them, 200 μl each of the bacterial suspension of transformed budding yeast TM182-BcOS1-I365S diluted in Glu-Ura-Leu medium as described above was dispensed and left at 30 ° C. for 67 hours. Cultured. On the other sheet, 200 μl of a suspension of transformed budding yeast TM182-BcOS1-I365S diluted in Gal-Ura-Leu medium as a control was dispensed and incubated at 30 ° C. for 67 hours. After incubation, the absorbance at 600 nm in each hole was measured with a microplate reader.
Table 2 shows the growth degrees of both the transformed budding yeast cultured in the presence of the compounds represented by the chemical formulas (1) to (7) (compounds (1) to (7)) and the budding yeast as a control thereof. It was. The degree of growth of both the transformed budding yeast and the control budding yeast were expressed as percentages, with the absorbance at 600 nm when the compound concentration was 0 ppm being 100. The degree of growth inhibition by each test substance of transformed budding yeast TM182-BcOS1-I365S is greater than the degree of growth inhibition by each test substance of transformed budding yeast AH22-BcOS1-I365S, and transformed budding yeast TM182-BcOS1-I365S Was confirmed to be a transformed cell with enhanced sensitivity to antibacterial active substances as compared to transformed budding yeast AH22-BcOS1-I365S.

Example 7 (Isolation of Osmotic Sensitive Histidine Kinase HIK1 Gene without Cell Transmembrane Region of Rice Blast Fungus)
First, total RNA was prepared from rice blast fungi. 100 mg of mycelia of rice blast fungus (Pyricularia grisea) P-37 grown on potato dextrose agar medium (PDA medium, Nissui Pharmaceutical Co., Ltd.) was scraped with a micro spatula in a liquid nitrogen and using a mortar and pestle And crushed. RNA was isolated from the frozen crushed powder using RNeasy Plant Mini Kit (QIAGEN). The frozen crushed powder was transferred to a 50 ml sample tube together with liquid nitrogen. After the liquid nitrogen had evaporated, the solution with 10 μl of mercaptoethanol per ml was added to the buffer RLC attached to the kit and stirred. . Furthermore, after the pulverized powder was well dispersed by pipetting several times, it was kept at 56 ° C. for 3 minutes. Thereafter, the solution containing the crushed powder was applied to a QIAshredder spin column attached to the kit and centrifuged at 8,000 × g for 2 minutes. The filtered supernatant was transferred to a new sample tube, 0.5 volume of 99.5% ethanol was added and mixed well by pipetting. This mixed solution was subjected to RNeasy mini spin column attached to the kit and centrifuged at 8,000 × g for 1 minute. The filtrate was discarded, 700 μl of buffer RW1 attached to the kit was added, and the mixture was centrifuged at 8,000 × g for 1 minute, and the filtrate was discarded. Furthermore, 500 μl of buffer RPE attached to the kit was added and centrifuged at 8,000 × g for 1 minute, and the filtrate was discarded. This operation was repeated twice. Finally, the upper filter portion was transferred to a new sample tube, subjected to 30 μl of RNase-free sterilized water attached to the kit, and centrifuged at 8,000 × g for 1 minute to elute the total RNA in the filtrate. This elution operation was repeated twice.
Next, cDNA was synthesized using the ThermoScript RT-PCR System (Invitrogen) using total RNA as a template. A solution in which 9.0 μl of total RNA was mixed with 1.0 μl of 50 mM Oligo (dT) ₂₀ attached to the kit and _2.0 μl of 10 mM dNTP Mix was treated at 65 ° C. for 5 minutes and rapidly cooled on ice. To this solution, add 4 μl of 5x cDNA Synthesis Buffer attached to the kit, 1 μl of 0.1M DTT, 1 μl of RNase OUT, 1 μl of ThermoScript RT, 1 μl of sterile distilled water, and react at 50 ° C. for 60 minutes. The reaction was stopped by heat treatment at 5 ° C. for 5 minutes. Further, 1 μl of RNaseH attached to the kit was added to this reaction solution, and reacted at 37 ° C. for 20 minutes to decompose the template RNA and synthesize cDNA.
Using this cDNA as a template, the HIK1 gene of rice blast fungus was amplified by PCR. PCR was carried out using the oligonucleotide consisting of the base sequence shown in SEQ ID NO: 18 and the oligonucleotide consisting of the base sequence shown in SEQ ID NO: 19 as primers to amplify the DNA having the base sequence shown in SEQ ID NO: 17. PCR reaction was carried out using KOD-Plus- (TOYOBO) at 94 ° C for 2 minutes, followed by 35 cycles of 94 ° C for 15 seconds, 55 ° C for 30 seconds, and 68 ° C for 6 minutes. Went under. The PCR reaction solution (50 μl) was 2 μl of the above cDNA, 5 μl of 10 × Buffer, 5 μl of 2 mM dNTPs, 2 μl of 25 mM MgSO ₄ , 1 μl of 10 μM oligonucleotide primer, 33 μl of sterile distilled water, KOD-Plus- Prepared by adding 1 μl. After the reaction, a part of the reaction solution was separated by 1.0% agarose gel electrophoresis, and it was confirmed that the HIK1 gene of about 4 kb was amplified by staining with ethidium bromide.

Example 8 (Construction of expression plasmid of HIK1 gene of rice blast fungus and preparation of transformed yeast)
The HIK1 gene of rice blast fungus was cloned into the cloning vector pBluescriptSKII (+). The reaction solution containing the HIK1 gene fragment prepared in Example 7 was purified using QIAquick PCR Purification Kit (QIAGEN) according to the attached manual. The purified HIK1 gene of about 4 kb was digested with restriction enzymes SpeI and HindIII, while the cloning vector pBluescriptSKII (+) (Stratagene) was digested with restriction enzymes SpeI and HindIII, followed by 1.0% agarose gel electrophoresis, respectively. And the gel part containing the target DNA was cut out. A gene fragment containing HIK1 digested with restriction enzymes SpeI and HindIII and a cloning vector digested with restriction enzymes SpeI and HindIII were collected from the gel using the QIAquick Gel Extraction Kit (QIAGEN) according to the attached manual. Using the Ligation Kit Ver.2 (TaKaRa), a plasmid pBlueHIK1 in which the HIK1 gene fragment was inserted between SpeI and HindIII of the multiple cloning site of the cloning vector was constructed. The nucleotide sequence of the obtained plasmid was subjected to a sequence reaction using BigDye terminator v3.0 Cycle Sequence FS Ready Reaction Kit (Applied Biosystems) according to the attached manual, and analyzed with a DNA sequencer (model 3100, Applied Biosystems). Sequencing reaction uses an oligonucleotide consisting of any one of the nucleotide sequences shown in SEQ ID NOs: 20 to 29 as a primer and repeats 35 cycles of 96 ° C for 10 seconds, 50 ° C for 5 seconds, and 60 ° C for 2 minutes Performed under conditions. As a result, the base sequence shown in SEQ ID NO: 17 was obtained, and it was confirmed that the plasmid pBlueH1K1 possesses the HIK1 gene.
Next, the HIK1 gene of rice blast fungus was inserted into the yeast-E. Coli shuttle vector p415ADH (ATCC87312). The plasmid pBlueHIK1 prepared as described above was digested with the restriction enzymes SpeI and HindIII, while the shuttle vector p415ADH (ATCC87312) was also digested with the restriction enzymes SpeI and HindIII and then separated by 1.0% agarose gel electrophoresis. The gel part containing DNA to be cut out was cut out. A gene fragment containing HIK1 digested with restriction enzymes SpeI and HindIII and a shuttle vector digested with restriction enzymes SpeI and HindIII were recovered from the gel using the QIAquick Gel Extraction Kit (QIAGEN) according to the attached manual. Plasmid pADHHIK1 in which the HIK1 gene fragment was inserted between SpeI and HindIII of the multiple cloning site of the shuttle vector was constructed using Ligation Kit Ver.2 (TaKaRa) according to the attached manual.
According to the method described in Geitz RD & Woods RA (1994) Molecular Genetics of Yeast: Practical Approaches ed. Johnson JA, Oxford University Press p124-134, the prepared expression plasmid pADHHIK1 The gene was introduced into the TM182 strain described in Maeda T et.al. (1994) Nature vol.369, p242-245. In the resulting transformed budding yeast, utilizing the loss of leucine auxotrophy, the transformed budding yeast strain AH22 (AH22-HIK1) was selected on a Glu-Leu agar medium, and transformed budding yeast strain TM182 (TM182 -HIK1) was selected on Gal-Ura-Leu agar medium. The obtained TM182-HIK1 was confirmed to grow even when transplanted to Glu-Ura-Leu medium.

Example 9 (Antimicrobial susceptibility test of transformed budding yeast)
The transformed budding yeast AH22-HIK1 prepared in Example 8 was cultured with shaking in Glu-Leu medium at 30 ° C. for 24 hours. As a control, the AH22 strain was similarly cultured with shaking in Glu medium at 30 ° C. for 24 hours. The absorbance at 600 nm of each of the grown transformed budding yeast cell suspensions was measured, and a cell suspension diluted with each medium so that the absorbance was 0.1 was prepared. Furthermore, the bacterial suspension of the transformed budding yeast AH22-HIK1 was diluted 50 times with Glu-Leu medium, and the bacterial suspension of the AH22 strain was diluted 50 times with Glu medium. A fungal suspension was prepared. A solution in which three kinds of compounds represented by chemical formulas (1) to (3) (compounds (1) to (3)) are dissolved in dimethyl sulfoxide (DMSO) to a concentration of 200 ppm each, ) And (5) two compounds (compounds (4) and (5)) dissolved in dimethyl sulfoxide (DMSO) to a concentration of 600 ppm, and chemical formulas (6) and Prepare a solution in which the two compounds shown in (7) (compounds (6) and (7)) are dissolved in dimethyl sulfoxide (DMSO) to a concentration of 20 ppm. As a result, two microplates were prepared in which DMSO and 1.0 μl each were dispensed into two holes. On one of them, 100 μl each of the suspension of transformed budding yeast AH22-HIK1 diluted as described above was dispensed and left to stand at 30 ° C. for 23 hours. On the other sheet, 100 μl of the cell suspension of the control yeast AH22 strain prepared as described above was dispensed and cultured at 30 ° C. for 27 hours. After incubation, the absorbance at 600 nm in each hole was measured with a microplate reader.
Similarly, the transformed budding yeast TM182-HIK1 prepared in Example 8 was cultured in a Glu-Ura-Leu medium at 30 ° C. for 24 hours. The absorbance at 600 nm of the grown bacterial suspension of transformed budding yeast was measured, and a bacterial suspension diluted with each medium so that the absorbance was 0.1 was prepared. Furthermore, the bacterial suspension of transformed Saccharomyces cerevisiae TM182-HIK1 was diluted 50-fold with Gal-Ura-Leu medium and a bacterial suspension diluted 50-fold with Glu-Ura-Leu medium. A fungal suspension was prepared. A solution in which three kinds of compounds represented by chemical formulas (1) to (3) (compounds (1) to (3)) are dissolved in dimethyl sulfoxide (DMSO) to a concentration of 200 ppm each, ) And (5) two compounds (compounds (4) and (5)) dissolved in dimethyl sulfoxide (DMSO) to a concentration of 600 ppm, and chemical formulas (6) and Prepare a solution in which the two compounds shown in (7) (compounds (6) and (7)) are dissolved in dimethyl sulfoxide (DMSO) to a concentration of 20 ppm. As a result, two microplates were prepared in which DMSO and 1.0 μl each were dispensed into two holes. On one of them, 100 μl each of the suspension of transformed Saccharomyces cerevisiae TM182-HIK1 diluted in Glu-Ura-Leu medium as described above was dispensed and incubated at 30 ° C. for 27 hours. . On the other side, as described above, 100 μl of the suspension of transformed Saccharomyces cerevisiae TM182-HIK1 diluted in Gal-Ura-Leu medium as a control was dispensed and left to stand at 30 ° C for 27 hours. did. After incubation, the absorbance at 600 nm in each hole was measured with a microplate reader.
Table 3 shows the growth levels of the transformed budding yeast and the control budding yeast for the compounds represented by the chemical formulas (1) to (7) (compounds (1) to (7)). The degree of growth of both the transformed budding yeast and the control budding yeast was expressed as a percentage, with the absorbance at 600 nm when the compound concentration was 0 ppm being 100. TM182-HIK1 growth inhibition by each test substance is greater than AH22-HIK1 growth inhibition by each test substance. It was confirmed to be a cell.

Example 10 (Amplification of osmotically sensitive histidine kinase gene fragments from other filamentous fungi)
(1) Preparation of total RNA of spinach wilt fungus Total RNA was isolated from spinach wilt fungus. 100 mg of mycelium of Fusarium oxysporum RJN1 grown on potato dextrose agar medium (PDA medium, Nissui Pharmaceutical) was scraped with a microspatula using a mortar and pestle in liquid nitrogen did. RNA was isolated from the frozen crushed powder according to the method described in Example 1 using RNeasy Plant Mini Kit (QIAGEN).

(2) Preparation of Total RNA of Wheat Leaf Blight Fungus Total RNA was isolated from wheat leaf blight fungus. Add spores of Mycospharella tritici St-8 strain grown on potato dextrose agar medium (PDA medium, Nissui Pharmaceutical) to 100 ml PD broth (DIFCO), and add 500 ml Erlenmeyer flask The culture was performed at 20 ° C. and 150 rpm for 4 days. The supernatant was removed by centrifuging 8 ml of the culture solution, and 300 mg of the live cell was transferred to a mortar and crushed with a pestle in liquid nitrogen. RNA was isolated from the frozen crushed powder according to the method described in Example 1 using RNeasy Plant Mini Kit (QIAGEN).

(3) Preparation of total RNA of rice mold blight fungus Total RNA was isolated from rice mold blight fungus. Add mycelium of rice rot fungus (Thanatephorus cucumeris) Rs-18 grown on potato dextrose agar medium (PDA medium, Nissui Pharmaceutical) to 100 ml PD broth (DIFCO), and add 500 ml Erlenmeyer flask The culture was statically cultured at 25 ° C. for 4 days. The supernatant was removed by centrifuging 8 ml of the culture solution, and the mycelium with a raw weight of 300 mg was transferred to a mortar and crushed with a pestle in liquid nitrogen. RNA was isolated from the frozen crushed powder according to the method described in Example 1 using RNeasy Plant Mini Kit (QIAGEN).

(4) Preparation of total RNA of tomato blight fungus Total RNA was isolated from tomato blight fungus. Rye culture medium (Phytophthora infestans) Pi-5 strain grown on rye medium agar (rye 60g, sucrose 15g, agar 20g / 1L), 20ml rye medium (rye 60g, sucrose 15g) / 1L), and cultured in a 300 ml Erlenmeyer flask at 20 ° C., 150 rpm for 5 days. The supernatant was removed by centrifuging 20 ml of the culture solution, and the microbial cells having a raw weight of 200 mg were transferred to a mortar and crushed with a pestle in liquid nitrogen. RNA was isolated from the frozen crushed powder according to the method described in Example 1 using RNeasy Plant Mini Kit (QIAGEN).

(5) Amplification of osmotic sensitive histidine kinase gene fragment by PCR Total RNA of rice blast fungus prepared in Example 7, total RNA of spinach wilt fungus prepared in Example 10 (1), or Wheat leaf blight fungus total RNA prepared in Example 10 (2), rice blight fungus total fungus prepared in Example 10 (3), prepared in Example 10 (4) Using total RNA of the tomato plague fungus, an osmotic pressure sensitive histidine kinase gene fragment was amplified.
First, cDNA was synthesized using ThermoScript RT-PCR System (Invitrogen) using each total RNA as a template. A solution was prepared by mixing 1.0 μl of 50 mM Okigo (dT) ₂₀ attached to the kit and _2.0 μl of 10 mM dNTP Mix with 4.0 μl of each total RNA and 5.0 μl of sterilized distilled water as described in Example 1. CDNA was synthesized according to the method.
PCR reaction was performed using each of these cDNAs as a template. As the primers, the primer pairs shown in Table 4 were used. The size of DNA expected to be amplified by PCR using each primer pair based on the base sequence shown in SEQ ID NO: 2 is shown in the table.

PCR reaction was carried out using KOD-Plus- (TOYOBO) at 94 ° C for 2 minutes, followed by incubation at 94 ° C for 15 seconds, then at 55 ° C for 30 seconds and then at 68 ° C for 1 minute. Amplification was repeated for 35 cycles. When primer pairs 1 to 6 were used, the incubation at 68 ° C. was 1 minute. When primer pairs 7-12 were used, the incubation at 68 ° C. was 5 minutes. When primer pairs 13 to 16 were used, the incubation at 68 ° C. was 3 minutes. The PCR reaction solution (25 μl) was 0.5 μl of the above cDNA, 2.5 μl of 10 × buffer, 2.5 μl of 8 mM dNTPs, 1.0 μl of 25 mM MgSO4, 0.5 μl of 10 μM oligonucleotide primer, 17 μl of sterile distilled water, KOD -Plus- was prepared by adding 0.5 μl. The PCR reaction solution after the reaction was analyzed by 1% or 4% agarose gel electrophoresis.
Using the rice blast fungus cDNA as a template, and using primer pairs 1, 2, 3, 4, 5 and 6, amplification of DNA of the expected size was observed. Amplification of DNA of the expected size was observed using primer pairs 2, 3, 7, 8, 9, 10, 11, 12 using cDNA of spinach wilt fungi as a template. Amplification of DNA of the expected size was observed using primer pairs 3, 5, 6, 13, 14, 15, and 16 using cDNA of wheat leaf blight fungi as a template. Amplification of DNA of the expected size was observed using primer pairs 2, 3, 5, and 6 using cDNA of rice mold blight fungus as a template. Using the cDNA of tomato wilt fungi as a template, amplification of DNA of the size expected using primer pairs 5 and 6 was observed.

Example 11 (Isolation of osmotically sensitive histidine kinase FoOS-1 gene having no transmembrane domain of spinach wilt fungus)
(1) Analysis of spinach wilt fungus FoOS-1 gene fragment The PCR reaction solution obtained by using primer pair 9 with the spinach wilt fungus cDNA amplified in Example 10 (5) as a template was used as a QIAquick. The amplified DNA was purified using PCR Purification Kit (QIAGEN) according to the attached instruction manual.
3′A addition was performed on the purified DNA using Ex Taq (TaKaRa). The reaction solution (20 μl) for 3′A addition was prepared by adding 15.3 μl of the purified PCR reaction solution, 2.0 μl of 10x buffer, 2.5 μl of 10 mM dNTPs, and 0.2 μl of Ex Taq. Was kept at 72 ° C. for 30 minutes.
The 3′A-added DNA and the pCR2.1-TOPO cloning vector (Invitrogen) were ligated according to the instruction manual attached to the cloning vector and then introduced into E. coli JM109 (TaKaRa). Plasmid DNA was purified from the obtained Escherichia coli transformant using QIAprep Spin Miniprep Kit (QIAGEN). BigDye Terminator v3.0 Cycle Sequencing Ready Reaction Kit (Applied Biosystems) using the obtained plasmid DNA as a template and an oligonucleotide consisting of any one of the nucleotide sequences shown in SEQ ID NOs: 28, 29, 45 to 48 A sequence reaction was carried out according to the instruction manual attached to the kit and analyzed with a DNA sequencer (Model 3100, Applied Biosystems). The sequence reaction was performed under amplification conditions of 96 cycles at 96 ° C. for 10 seconds, 50 ° C. for 5 seconds, and 60 ° C. for 2 minutes for one cycle, and this was repeated 35 cycles. As a result, the base sequence represented by base numbers 663 to 3534 of the base sequence represented by SEQ ID NO: 42 was read.

(2) Analysis of the full length FoOS-1 gene of spinach wilt fungus
Using the SMART RACE cDNA Amplification Kit (CLONTECH), DNA extending 5 ′ upstream from the base number 663 of the base sequence shown in SEQ ID NO: 42 was cloned according to the instruction manual attached to the kit. Specifically, 3 μl (230 ng) of the total RNA prepared in Example 10 (1) was mixed with 1.0 μl of CDS-primer attached to the kit and 1.0 μl of SMART IIA Oligo to prepare a reaction solution. The mixture was incubated at 70 ° C. for 2 minutes and then kept on ice for 2 minutes. To the reaction solution, 2 μl of 5 × First-Strand buffer attached to the kit, 1 μl of 20 mM DTT, 1 μl of 10 mM dNTP Mix and 1 μl of PowerScript Reverse Transcriptase were added and mixed, and incubated at 42 ° C. for 1.5 hours. After adding 100 μl of Tricine-EDTA buffer attached to the kit to the reaction solution after the incubation, the mixture was incubated at 72 ° C. for 7 minutes to prepare 5 ′ RACE ready cDNA. Using this 5 ′ RACE ready cDNA as a template, PCR was performed to amplify the 5 ′ upstream region. The PCR reaction solution was mixed with 2.5 μl of 5 ′ RACE ready cDNA, 5.0 μl of 10x Advantage 2 buffer attached to the kit, 1.0 μl of 10 mM dNTP Mix, and 1.0 μl of 50x Advantage 2 Polymerase Mix. 5.0 μl of 10 × Universal Primer A Mix attached to the kit and 1.0 μl of 10 μM oligonucleotide solution consisting of the base sequence shown in SEQ ID NO: 43 were added, and the total volume was made 50 μl using sterile distilled water. . This reaction solution was kept at 94 ° C. for 5 seconds and then at 72 ° C. for 2 minutes as one cycle, and this was repeated 5 cycles, followed by further incubation at 94 ° C. for 5 seconds, then 70 ° C. for 10 seconds, and then at 72 ° C. for 2 minutes. The cycle was repeated 5 cycles, and further, this was repeated 25 cycles with 94 ° C. for 5 seconds, then 68 ° C. for 10 seconds and then 72 ° C. for 2 minutes, and then kept at 72 ° C. for 7 minutes. The PCR reaction solution and the pCR2.1-TOPO cloning vector (Invitrogen) were ligated according to the instruction manual attached to the cloning vector, and then introduced into E. coli JM109 (TaKaRa). Plasmid DNA was purified from the obtained Escherichia coli transformant using QIAprep Spin Miniprep Kit (QIAGEN). The obtained plasmid DNA was used as a template, and the nucleotide sequence was analyzed according to the method described in Example 11 (1) using primers consisting of the nucleotide sequences represented by SEQ ID NOs: 29, 49 and 54. As a result, the base sequence represented by base numbers 1 to 662 of the base sequence represented by SEQ ID NO: 42 was read.
Furthermore, DNA extending to the 3 ′ downstream region from base number 3534 of the base sequence represented by SEQ ID NO: 42 was cloned. Specifically, 3 μl (230 ng) of the total RNA prepared in Example 10 (1) was mixed with 1.0 μl of CDS-primer attached to the kit and 1.0 μl of sterilized distilled water. After being kept warm for 1 minute, it was kept warm on ice for 2 minutes. Using the reaction solution, 3 ′ RACE ready cDNA was prepared in the same manner as 5 ′ RACE ready cDNA preparation. Using this 3 ′ RACE ready cDNA as a template, PCR was performed to amplify the 3 ′ downstream region. The PCR reaction solution was mixed with 2.5 μl of 5 ′ RACE ready cDNA, 5.0 μl of 10x Advantage 2 buffer attached to the kit, 1.0 μl of 10 mM dNTP Mix, and 1.0 μl of 50x Advantage 2 Polymerase Mix. 5.0 μl of 10 × Universal Primer A Mix attached to the kit and 1.0 μl of 10 μM oligonucleotide solution consisting of the base sequence shown in SEQ ID NO: 42 were added, and the total volume was adjusted to 50 μl using sterile distilled water. This reaction solution was kept at 94 ° C. for 5 seconds and then at 72 ° C. for 2 minutes as one cycle, and this was repeated 5 cycles, followed by further incubation at 94 ° C. for 5 seconds, then 70 ° C. for 10 seconds, and then at 72 ° C. for 2 minutes. The cycle was repeated 5 cycles, and further, this was repeated 25 cycles with 94 ° C. for 5 seconds, then 68 ° C. for 10 seconds and then 72 ° C. for 2 minutes, and then kept at 72 ° C. for 7 minutes. The PCR reaction solution and the pCR2.1-TOPO cloning vector (Invitrogen) were ligated according to the instruction manual attached to the vector, and then introduced into E. coli JM109 (TaKaRa). Plasmid DNA was purified from the obtained Escherichia coli transformant using QIAprep Spin Miniprep Kit (QIAGEN). The obtained plasmid DNA was used as a template, and the nucleotide sequence was analyzed according to the method described in Example 11 (1) using primers consisting of the nucleotide sequences represented by SEQ ID NOs: 29, 50 and 54. As a result, the base sequence represented by base numbers 3535 to 3882 of the base sequence represented by SEQ ID NO: 42 was read.
As a result of linking all the analyzed base sequences, the base sequence represented by SEQ ID NO: 42 was obtained. The base sequence represented by SEQ ID NO: 42 consisted of 3882 bases (including a stop codon), and was a base sequence encoding 1293 amino acid residues (SEQ ID NO: 41). The molecular weight of the protein having the amino acid sequence represented by SEQ ID NO: 41 was calculated to be 141818 Da.

(3) Isolation of the full-length gene of spinach wilt fungus FoOS-1 Using the 5 'RACE ready cDNA prepared in Example 11 (2) as a template, the FoOS-1 gene of spinach wilt fungi was amplified by PCR. . A PCR reaction was carried out using the oligonucleotide consisting of the base sequence shown by SEQ ID NO: 52 and the oligonucleotide consisting of the base sequence shown by SEQ ID NO: 53 as primers to amplify the DNA having the base sequence shown by SEQ ID NO: 42. PCR reaction was carried out using KOD-Plus- (TOYOBO) at 94 ° C for 2 minutes, followed by incubation at 94 ° C for 15 seconds, then at 55 ° C for 30 seconds and then at 68 ° C for 6 minutes as one cycle. Amplification was repeated for 35 cycles. The PCR reaction solution (50 μl) was 2.5 μl of 5 ′ RACE ready cDNA, 5.0 μl of 10 × buffer, 5.0 μl of 2 mM dNTPs, 2.0 μl of 25 mM MgSO4, 1.0 μl of 10 μM oligonucleotide primer, and sterile distilled water. 32.5 μl and KOD-Plus- were prepared by adding 1.0 μl. After the reaction, a part of the PCR reaction solution was separated by 1% agarose gel electrophoresis and stained with ethidium bromide to confirm that about 4 kb of FoOS-1 gene was amplified.

Example 12 (Construction of FoOS-1 gene expression plasmid of spinach wilt fungus and production of transformed budding yeast)
First, the FoOS-1 gene of spinach wilt fungus was subcloned into the pCR2.1-TOPO cloning vector (Invitrogen). The reaction solution containing about 4 kb of FoOS-1 gene prepared in Example 11 (3) was purified using QIAquick PCR Purification Kit (QIAGEN) according to the manual attached to the kit. 3′A addition was performed on the purified 4 kb FoOS-1 gene according to the method described in Example 11 (1). The plasmid pCRFoOS1 was constructed by ligating the FoOS-1 gene added with 3′A and the pCR2.1-TOPO cloning vector (Invitrogen) according to the instruction manual attached to the cloning vector. The nucleotide sequence of the obtained plasmid was analyzed according to the method described in Example 11 (1). As a primer, an oligonucleotide consisting of any one of the base sequences shown in SEQ ID NOs: 29, 43 to 51, and 54 was used. As a result, the base sequence shown in SEQ ID NO: 42 was obtained, and it was confirmed that the plasmid pCRFoOS1 contains the FoOS-1 gene.
The FoOS-1 gene contained in the plasmid pCRFoOS1 prepared in this way was cloned into the yeast-E. Coli shuttle vector p415ADH to construct an expression plasmid. Plasmid pCRFoOS1 was digested with restriction enzymes SpeI and PstI, while shuttle vector p415ADH was also digested with restriction enzymes SpeI and PstI. After separating them by 0.8% agarose gel electrophoresis, the FoOS-1 gene fragment digested with restriction enzymes SpeI and PstI and the shuttle vector p415ADH digested with restriction enzymes SpeI and PstI were excised, and QIAquick Gel Extraction Kit (QIAGEN The FoOS-1 gene fragment digested with the restriction enzymes SpeI and PstI and the shuttle vector digested with the restriction enzymes SpeI and PstI were recovered from the gel according to the manual attached using Using Ligation Kit Ver.2 (TaKaRa), construct the expression plasmid pADHFoOS1 by inserting the FoOS-1 gene between SpeI and PstI of the multicloning site of the shuttle vector according to the manual attached to the kit. did. The base sequence of the obtained expression plasmid was analyzed according to the method described in Example 11 (1). As a primer, an oligonucleotide having a base sequence represented by SEQ ID NOs: 43 to 53 was used. As a result, the base sequence shown in SEQ ID NO: 42 was obtained, and it was confirmed that the expression plasmid pADHFoOS1 possesses the FoOS-1 gene.
The prepared expression plasmid pADHFoOS1 was introduced into the budding yeast strain AH22 and TM182 according to the method described in Example 2. In the obtained transformed budding yeast, the auxotrophy of leucine disappears, and the transformed budding yeast AH22 strain (AH22-FoOS1) is selected on the Glu-Leu agar medium, and the transformed budding yeast strain TM182 ( TM182-FoOS1) was selected on Gal-Ura-Leu agar medium. The obtained TM182-FoOS1 was confirmed to grow even when transplanted to Glu-Ura-Leu medium.

Example 13 (Antimicrobial susceptibility test of transformed budding yeast TM182-FoOS1)
The transformed budding yeast AH22-FoOS1 prepared in Example 12 was cultured with shaking at 30 ° C. for 18 hours in a Glu-Leu medium. As a control, the AH22 strain was similarly cultured with shaking in Glu medium at 30 ° C. for 18 hours. The absorbance at 600 nm in each of the grown transformed budding yeast cell suspensions was measured, and a cell suspension diluted with sterilized distilled water was prepared so that the absorbance was 0.1. Furthermore, a bacterial suspension in which the bacterial suspension of transformed budding yeast AH22-FoOS1 is diluted 50 times with Glu-Leu medium; a bacterial suspension in which AH22 strain is diluted 50 times with Glu medium; Was prepared.
A solution in which three kinds of compounds represented by chemical formulas (1) to (3) (compounds (1) to (3)) are dissolved in dimethyl sulfoxide (DMSO) so as to have a concentration of 600 ppm each, ) And (5) two compounds (compounds (4) and (5)) dissolved in dimethyl sulfoxide (DMSO) to a concentration of 2000 ppm, and chemical formulas (6) and (6) Prepare a solution in which the two compounds shown in (7) (compounds (6) and (7)) are dissolved in dimethyl sulfoxide (DMSO) to a concentration of 20 ppm. As a result, two microplates were prepared in which DMSO and 1.0 μl each were dispensed into two holes. On one of them, 100 μl each of the suspension of transformed budding yeast AH22-FoOS1 diluted and prepared as described above was dispensed and statically cultured at 30 ° C. for 26.5 hours. On the other sheet, 100 μl of the cell suspension of the control yeast strain AH22 diluted as described above was dispensed and cultured at 30 ° C. for 24.5 hours. After incubation, the absorbance at 600 nm in each hole was measured with a microplate reader.
Similarly, the transformed budding yeast TM182-FoOS1 prepared in Example 12 was cultured in Glu-Ura-Leu medium at 30 ° C. for 18 hours. The absorbance at 600 nm of the bacterial suspension of the grown transformed budding yeast was measured, and a bacterial suspension diluted with sterile distilled water was prepared so that the absorbance was 0.1. Furthermore, a bacterial suspension in which transformed budding yeast TM182-FoOS1 was diluted 50-fold with Glu-Ura-Leu medium and a bacterial suspension diluted 50-fold with Gal-Ura-Leu medium as a control. Prepared.
A solution in which three kinds of compounds represented by chemical formulas (1) to (3) (compounds (1) to (3)) are dissolved in dimethyl sulfoxide (DMSO) so as to have a concentration of 600 ppm each, ) And (5) two compounds (compounds (4) and (5)) dissolved in dimethyl sulfoxide (DMSO) to a concentration of 2000 ppm, and chemical formulas (6) and (6) Prepare a solution in which the two compounds shown in (7) (compounds (6) and (7)) are dissolved in dimethyl sulfoxide (DMSO) to a concentration of 20 ppm. As a result, two microplates were prepared in which DMSO and 2.0 μl were dispensed into each hole. On one of them, 100 μl each of the suspension of transformed budding yeast TM182-FoOS1 diluted and prepared in Glu-Ura-Leu medium as described above was dispensed and incubated at 30 ° C. for 25 hours. . On the other side, as described above, 100 μl each of the suspension of transformed budding yeast TM182-FoOS1 diluted in Gal-Ura-Leu medium as a control was dispensed and statically cultured at 30 ° C. for 51 hours. did. After incubation, the absorbance at 600 nm in each hole was measured with a microplate reader.
Table 5 shows the degree of growth of each transformed budding yeast for the compounds represented by chemical formulas (1) to (7) (compounds (1) to (7)). The degree of growth of the transformed budding yeast was expressed as a percentage relative to the absorbance at 600 nm when the compound concentration was 0 ppm as 100. The degree of growth inhibition by each test substance of transformed budding yeast TM182-FoOS1 is greater than the degree of growth inhibition by each test substance of transformed budding yeast AH22-FoOS1, and transformed budding yeast TM182-FoOS1 -It was confirmed that the transformed cells had enhanced sensitivity to antibacterial active substances compared with -FoOS1.

Example 14 (Isolation of osmotically sensitive histidine kinase StOS-1 gene having no transmembrane domain of wheat leaf blight fungus)
(1) Analysis of Wheat Leaf Blight Fungus StOS-1 Gene Fragment A PCR reaction solution obtained in Example 10 (4) using a cDNA of wheat leaf blight fungus cDNA as a template and using primer pair 16 was used. The product was purified using a QIAquick PCR Purification Kit (QIAGEN) according to the instruction manual attached to the kit. The amplified DNA obtained by purification was subjected to 3′A addition according to the method described in Example 11 (1). The 3′A-added DNA and the pCR2.1-TOPO cloning vector (Invitrogen) were ligated according to the instruction manual attached to the cloning vector, and then introduced into E. coli JM109 (TaKaRa).
A DNA fragment was amplified from the obtained Escherichia coli transformant by colony PCR using Ex Taq HS (TaKaRa). The PCR reaction solution (15 μl) is composed of 1.5 μl of 10 × buffer, 2.25 μl of 10 mM dNTPs, 0.15 μl of Ex Taq HS, 10 μM oligonucleotide solution consisting of the base sequence represented by SEQ ID NO: 66 and the base represented by SEQ ID NO: 67 0.4 μl each of 10 μM solution of oligonucleotide consisting of sequence and 10.3 μl of sterilized distilled water were mixed and prepared by adding a part of E. coli transformant colony. This reaction solution was kept at 97 ° C. for 2 minutes, and then kept at 97 ° C. for 15 seconds, then at 55 ° C. for 15 seconds and further at 72 ° C. for 3 minutes. The incubated PCR reaction solution was purified using a QIAquick PCR purification Kit (QIAGEN) according to the instruction manual attached to the kit. The base sequence was analyzed according to the method described in Example 11 (1) using the PCR amplified fragment obtained by purification as a template and using the oligonucleotide consisting of the base sequences shown in SEQ ID NOs: 29 and 54 as primers. As a result, the base sequence represented by base numbers 2241 to 3603 of the base sequence represented by SEQ ID NO: 56 was read.

(2) Analysis of the full length StOS-1 gene of wheat leaf blight fungus
Using the SMART RACE cDNA Amplification Kit (CLONTECH), DNA extending 5 ′ upstream from the base number 2241 of the base sequence represented by SEQ ID NO: 56 was cloned according to the instruction manual attached to the kit. Specifically, the reaction mixture was prepared by mixing 3 μl (230 ng) of the total RNA prepared in Example 10 (2), 1.0 μl of CDS-primer attached to the kit and 1.0 μl of SMART IIA Oligo. The mixture was kept at 70 ° C. for 2 minutes, and then kept on ice for 2 minutes. To the reaction solution, 2 μl of 5 × First-Strand buffer attached to the kit, 1 μl of 20 mM DTT, 1 μl of 10 mM dNTP Mix and 1 μl of PowerScript Reverse Transcriptase were added and mixed, and incubated at 42 ° C. for 1.5 hours. After adding 100 μl of Tricine-EDTA buffer attached to the kit to the reaction solution after the incubation, the mixture was incubated at 72 ° C. for 7 minutes to prepare 5 ′ RACE ready cDNA. Using this 5 ′ RACE ready cDNA as a template, PCR was performed to amplify the 5 ′ upstream region using KOD-plus- (TOYOBO). The PCR reaction solution is 2.5 μl of 5 ′ RACE ready cDNA, 5.0 μl of 10 × buffer, 5.0 μl of 2 mM dNTPs, 2.0 μl of 25 mM MgSO4, and 1.0 μl of KOD-Plus-, and the primers are attached to the kit. Furthermore, 5.0 μl of 10 × Universal Primer A Mix and 1.0 μl of 10 μM oligonucleotide solution consisting of the base sequence shown in SEQ ID NO: 43 were added, and the total volume was adjusted to 50 μl using sterilized distilled water. This reaction solution was kept at 94 ° C. for 2 minutes, and further, this was repeated 35 times with one cycle of 94 ° C. for 15 seconds and then 68 ° C. for 5 minutes. The PCR reaction solution was purified using a QIAquick PCR Purification Kit (QIAGEN) according to the instruction manual attached to the kit, and the purified DNA was purified according to the method described in Example 11 (1). 3'A addition was performed. The 3′A-added DNA and the pCR2.1-TOPO cloning vector (Invitrogen) were ligated according to the instruction manual attached to the cloning vector, and then introduced into E. coli JM109 (TaKaRa). Plasmid DNA was purified from the obtained Escherichia coli transformant using QIAprep Spin Miniprep Kit (QIAGEN). The obtained plasmid DNA was used as a template, and the nucleotide sequence was analyzed according to the method described in Example 11 (1) using primers consisting of nucleotide sequences represented by SEQ ID NOs: 29, 54 and 59 to 61. As a result, the base sequence represented by base numbers 1 to 2240 of the base sequence represented by SEQ ID NO: 56 was read.
Furthermore, DNA extending to the 3 ′ downstream region from the base number 3603 of the base sequence represented by SEQ ID NO: 56 was cloned. Specifically, 3 μl (230 ng) of the total RNA prepared in Example 10 (2) was mixed with 1.0 μl of CDS-primer attached to the kit and 1.0 μl of sterilized distilled water. After being kept warm for 1 minute, the temperature was kept on ice for 2 minutes. Using the reaction solution, 3 ′ RACE ready cDNA was prepared in the same manner as 5 ′ RACE ready cDNA preparation. Using this 3 ′ RACE ready cDNA as a template, PCR was performed to amplify the 3 ′ downstream region. The PCR reaction solution was mixed with 2.5 μl of 5 ′ RACE ready cDNA, 5.0 μl of 10x Advantage 2 buffer attached to the kit, 1.0 μl of 10 mM dNTP Mix, and 1.0 μl of 50x Advantage 2 Polymerase Mix. 5.0 μl of 10 × Universal Primer A Mix attached to the kit and 1.0 μl of 10 μM solution of the oligonucleotide consisting of the base sequence represented by SEQ ID NO: 58 were added, and the total volume was made 50 μl using sterile distilled water. . This reaction solution was kept at 94 ° C. for 5 seconds and then at 72 ° C. for 4 minutes as one cycle, and this was repeated 5 cycles. Further, 94 ° C. for 5 seconds, 70 ° C. for 10 seconds, then 72 ° C. for 4 minutes. This was repeated as 5 cycles, and further, this was repeated 25 cycles with 94 ° C. for 5 seconds, then 68 ° C. for 10 seconds, then 72 ° C. for 4 minutes, and then kept at 72 ° C. for 7 minutes. The PCR reaction solution and the pCR2.1-TOPO cloning vector (Invitrogen) were ligated according to the instruction manual attached to the cloning vector, and then introduced into E. coli JM109 (TaKaRa). Plasmid DNA was purified from the obtained Escherichia coli transformant using QIAprep Spin Miniprep Kit (QIAGEN). The obtained plasmid DNA was used as a template, and the nucleotide sequence was analyzed according to the method described in Example 11 (1) using primers consisting of the nucleotide sequences represented by SEQ ID NOs: 29 and 54. As a result, the base sequence represented by base numbers 3604 to 3924 of the base sequence represented by SEQ ID NO: 56 was read.
As a result of linking all the analyzed base sequences, the base sequence represented by SEQ ID NO: 56 was obtained. The base sequence represented by SEQ ID NO: 56 consisted of 3924 bases (including a stop codon), and was a base sequence encoding 1307 amino acid residues (SEQ ID NO: 55). The molecular weight of the protein having the amino acid sequence represented by SEQ ID NO: 55 was calculated to be 143276 Da.

(3) Isolation of full-length StOS-1 gene of wheat leaf blight fungus StOS-1 gene of wheat leaf blight fungus by PCR using 5 'RACE ready cDNA prepared in Example 14 (2) as a template Amplified. By performing a PCR reaction according to the method described in Example 11 (3) using the oligonucleotide having the base sequence represented by SEQ ID NO: 64 and the oligonucleotide having the base sequence represented by SEQ ID NO: 65 as primers, SEQ ID NO: 56 A DNA having the base sequence indicated by was amplified.
PCR reaction was carried out using KOD-Plus- (TOYOBO) at 94 ° C for 2 minutes, followed by incubation at 94 ° C for 15 seconds, then at 55 ° C for 30 seconds and then at 68 ° C for 6 minutes as one cycle. Amplification was repeated for 35 cycles. The PCR reaction solution (50 μl) was 2.5 μl of 5 ′ RACE ready cDNA, 5.0 μl of 10 × buffer, 5.0 μl of 2 mM dNTPs, 2.0 μl of 25 mM MgSO4, 1.0 μl of 10 μM oligonucleotide primer, and sterile distilled water. 32.5 μl and KOD-Plus- were prepared by adding 1.0 μl.
A part of the PCR reaction solution after the reaction was separated by 1% agarose gel electrophoresis, and it was confirmed that the StOS-1 gene of about 4 kb was amplified by staining with ethidium bromide.

Example 15 (Construction of expression plasmid of wheat leaf blight fungus StOS-1 gene and production of transformed budding yeast)
First, the wheat leaf blight fungus StOS-1 gene was subcloned into the pCR2.1-TOPO cloning vector (Invitrogen). The reaction solution containing the StOS-1 gene prepared in Example 14 (3) was purified using QIAquick PCR Purification Kit (QIAGEN) according to the manual attached to the kit. According to the method described in Example 11 (1), 3′A addition was performed on the purified DNA of about 4 kb. The plasmid pCRStOS1 was constructed by ligating the 3′A-added DNA and the pCR2.1-TOPO cloning vector (Invitrogen) according to the instruction manual attached to the cloning vector. The nucleotide sequence of the obtained plasmid was analyzed according to the method described in Example 11 (1). As a primer, an oligonucleotide consisting of any one of the base sequences shown in SEQ ID NOs: 29, 54 and 58 to 63 was used. As a result, the base sequence shown in SEQ ID NO: 56 was obtained, and it was confirmed that the plasmid pCRStOS1 contains the StOS-1 gene.
An expression plasmid was constructed by cloning the StOS-1 gene contained in the thus prepared plasmid pCR StOS1 into the yeast-E. Coli shuttle vector p415ADH. Plasmid pCRStOS1 was digested with restriction enzymes SpeI and HindIII, while shuttle vector p415ADH was also digested with restriction enzymes SpeI and HindIII. These were separated by 0.8% agarose gel electrophoresis, respectively, and then the gel portion containing the StOS-1 gene fragment digested with restriction enzymes SpeI and HindIII and the shuttle vector p415ADH digested with restriction enzymes SpeI and HindIII was excised. Using the QIAquick Gel Extraction Kit (QIAGEN), collect the StOS-1 gene fragment digested with the restriction enzymes SpeI and HindIII and the shuttle vector digested with the restriction enzymes SpeI and HindIII from the gel according to the manual attached to the kit. did. Using Ligation Kit Ver.2 (TaKaRa), construct the expression plasmid pADHStOS1 by inserting the StOS-1 gene between SpeI and HindIII of the multicloning site of the shuttle vector according to the manual attached to the kit. did. The base sequence of the obtained expression plasmid was analyzed according to the method described in Example 11 (1). As a primer, an oligonucleotide consisting of any one of the base sequences shown in SEQ ID NOs: 58 to 65 was used. As a result, the base sequence shown in SEQ ID NO: 56 was obtained, and it was confirmed that the expression plasmid pADHFoOS1 was an expression plasmid carrying the StOS-1 gene.
The prepared expression plasmid pADH StOS1 was introduced into the budding yeast strains AH22 and TM182 according to the method described in Example 2. In the resulting transformed budding yeast, the auxotrophy of leucine disappears, and the transformed budding yeast AH22 strain (AH22-StOS1) is selected on the Glu-Leu agar medium, and the transformed budding yeast strain TM182 (TM182 -StOS1) was selected on Gal-Ura-Leu agar medium. The obtained TM182-StOS1 was confirmed to grow even when transplanted to Glu-Ura-Leu medium.

Example 16 (Antimicrobial susceptibility test of transformed budding yeast TM182-StOS1)
The transformed budding yeast AH22-StOS1 prepared in Example 15 was cultured with shaking at 30 ° C. for 18 hours in a Glu-Leu medium. As a control, the AH22 strain was similarly cultured with shaking in Glu medium at 30 ° C. for 18 hours. The absorbance at 600 nm in each of the grown transformed budding yeast cell suspensions was measured, and a cell suspension diluted with sterilized distilled water was prepared so that the absorbance was 0.1. Furthermore, a bacterial suspension in which transformed budding yeast AH22-StOS1 was diluted 50 times with Glu-Leu medium and a bacterial suspension in which AH22 strain was diluted 50 times with Glu medium were prepared.
A solution in which three kinds of compounds represented by chemical formulas (1) to (3) (compounds (1) to (3)) are dissolved in dimethyl sulfoxide (DMSO) to a concentration of 6 ppm each, ) And (5), two compounds (compounds (4) and (5)) dissolved in dimethyl sulfoxide (DMSO) to a concentration of 2000 ppm, and chemical formula (6) And a solution in which the two compounds shown in (7) (compounds (6) and (7)) are each dissolved in dimethyl sulfoxide (DMSO) to a concentration of 20 ppm, and the compound DMSO solution is prepared. As a control, two microplates were prepared in which DMSO was dispensed at two locations (ie, two wells), each with 1.0 μl in each well. On one of them, 100 μl each of the suspension of transformed budding yeast AH22-StOS1 diluted and prepared as described above was dispensed and statically cultured at 30 ° C. for 28 hours. On the other sheet, 100 μl of the cell suspension of the control yeast strain AH22 diluted as described above was dispensed and cultured at 30 ° C. for 24.5 hours. After incubation, the absorbance at 600 nm in each hole was measured with a microplate reader.
Similarly, the transformed budding yeast TM182-StOS1 prepared in Example 2 was cultured in a Glu-Ura-Leu medium at 30 ° C. for 18 hours. The absorbance at 600 nm of the bacterial suspension of the grown transformed budding yeast was measured, and a bacterial suspension diluted with sterile distilled water was prepared so that the absorbance was 0.1. Furthermore, a bacterial suspension in which transformed budding yeast TM182-StOS1 was diluted 50-fold with Glu-Ura-Leu medium and a bacterial suspension diluted 50-fold with Gal-Ura-Leu medium as a control. Prepared.
A solution in which three kinds of compounds represented by chemical formulas (1) to (3) (compounds (1) to (3)) are dissolved in dimethyl sulfoxide (DMSO) to a concentration of 6 ppm each, ) And (5), two compounds (compounds (4) and (5)) dissolved in dimethyl sulfoxide (DMSO) to a concentration of 2000 ppm, and chemical formula (6) And a solution in which the two compounds shown in (7) (compounds (6) and (7)) are each dissolved in dimethyl sulfoxide (DMSO) to a concentration of 20 ppm, and the compound DMSO solution is prepared. As a control, two microplates were prepared in which DMSO was dispensed in two locations (ie, two wells), each with 2.0 μl in each hole. On one of them, 100 μl each of the suspension of transformed Saccharomyces cerevisiae TM182-StOS1 diluted in Glu-Ura-Leu medium as described above was dispensed and incubated at 30 ° C. for 26.5 hours. . On the other side, as described above, 100 μl of the suspension of transformed budding yeast TM182-StOS1 diluted in Gal-Ura-Leu medium as a control was dispensed as above, and incubated at 30 ° C for 49.5 hours. did. After incubation, the absorbance at 600 nm in each hole was measured with a microplate reader.
Table 6 shows the degree of growth of each transformed budding yeast for the compounds represented by chemical formulas (1) to (7) (compounds (1) to (7)). The degree of growth of the transformed budding yeast was expressed as a percentage relative to the absorbance at 600 nm when the compound concentration was 0 ppm as 100. The degree of growth inhibition by each test substance of transformed budding yeast TM182-StOS1 is greater than the degree of growth inhibition by each test substance of transformed budding yeast AH22-StOS1, and the transformed budding yeast TM182-StOS1 -It was confirmed that the transformed cells had enhanced sensitivity to antibacterial active substances compared to StOS1.

Example 17 (Isolation of Osmotic Sensitive Histidine Kinase RsOS-1 Gene without Cell Transmembrane Region of Rice Blight Fungus)
(1) Analysis of rice mold blight fungus RsOS-1 gene fragment A PCR reaction solution obtained by using primer pair 3 with the rice mold blight fungus cDNA amplified in Example 10 (5) as a template was used. The product was purified using a QIAquick PCR Purification Kit (QIAGEN) according to the instruction manual attached to the kit. The amplified DNA obtained by purification was subjected to 3′A addition according to the method described in Example 11 (1). The 3′A-added DNA and the pCR2.1-TOPO cloning vector (Invitrogen) were ligated according to the instruction manual attached to the cloning vector, and then introduced into E. coli JM109 (TaKaRa).
A DNA fragment was amplified from the obtained Escherichia coli transformant by colony PCR using Ex Taq HS (TaKaRa). The PCR reaction solution (15 μl) is composed of 1.5 μl of 10 × buffer, 2.25 μl of 10 mM dNTPs, 0.15 μl of Ex Taq HS, 10 μM oligonucleotide solution consisting of the base sequence represented by SEQ ID NO: 28 and the base represented by SEQ ID NO: 29 0.4 μl each of 10 μM solution of oligonucleotide consisting of sequence and 10.3 μl of sterilized distilled water were mixed and prepared by adding a part of E. coli transformant colony. This reaction solution was kept at 97 ° C. for 2 minutes, and then kept at 97 ° C. for 15 seconds, then at 55 ° C. for 15 seconds and further at 72 ° C. for 3 minutes. The incubated PCR reaction solution was purified using a QIAquick PCR purification Kit (QIAGEN) according to the instruction manual attached to the kit. The nucleotide sequence was analyzed according to the method described in Example 11 (1) using the PCR amplified fragment obtained by purification as a template and using the oligonucleotide consisting of the nucleotide sequences represented by SEQ ID NOs: 28 and 29 as primers. As a result, the base sequence represented by base numbers 2838 to 3165 of the base sequence represented by SEQ ID NO: 69 was read.

(2) Analysis of the full-length rice filamentous fungus RsOS-1 gene
Using the SMART RACE cDNA Amplification Kit (CLONTECH), DNA extending 3 ′ downstream from the base number 3165 of the base sequence represented by SEQ ID NO: 69 was cloned according to the instruction manual attached to the kit. Specifically, 3 μl (253 ng) of the total RNA prepared in Example 10 (3) was mixed with 1.0 μl of CDS-primer attached to the kit and 1.0 μl of water to prepare a reaction solution, After incubating at 70 ° C. for 2 minutes, the mixture was kept on ice for 2 minutes. To the reaction solution, 2 μl of 5 × First-Strand buffer attached to the kit, 1 μl of 20 mM DTT, 1 μl of 10 mM dNTP Mix and 1 μl of PowerScript Reverse Transcriptase were added and mixed, and incubated at 42 ° C. for 1.5 hours. After adding 100 μl of Tricine-EDTA buffer attached to the kit to the reaction solution after the incubation, the mixture was incubated at 72 ° C. for 7 minutes to prepare 3 ′ RACE ready cDNA. Using this 3 ′ RACE ready cDNA as a template, PCR was performed to amplify the 3 ′ downstream region using KOD-plus- (TOYOBO). The PCR reaction solution is 2.5 μl of 3 ′ RACE ready cDNA, 5.0 μl of 10 × buffer, 5.0 μl of 2 mM dNTPs, 2.0 μl of 25 mM MgSO4, and 1.0 μl of KOD-Plus-. Furthermore, 5.0 μl of 10 × Universal Primer A Mix and 1.0 μl of 10 μM oligonucleotide solution consisting of the base sequence shown in SEQ ID NO: 70 were added, and the total volume was adjusted to 50 μl using sterilized distilled water. This reaction solution was kept at 94 ° C. for 2 minutes, and further, this was repeated 35 times with one cycle of 94 ° C. for 15 seconds and then 68 ° C. for 6 minutes. The PCR reaction solution is purified using the QIAquick PCR Purification Kit (QIAGEN) according to the instruction manual attached to the kit, and the purified DNA is purified according to the method described in Example 11 (1). 3'A addition was performed. The 3′A-added DNA and the pCR2.1-TOPO cloning vector (Invitrogen) were ligated according to the instruction manual attached to the cloning vector, and then introduced into E. coli JM109 (TaKaRa). Plasmid DNA was purified from the obtained Escherichia coli transformant using QIAprep Spin Miniprep Kit (QIAGEN). The obtained plasmid DNA was used as a template, and the nucleotide sequence was analyzed according to the method described in Example 11 (1) using primers consisting of nucleotide sequences represented by SEQ ID NOs: 28, 29 and 73 to 76. As a result, the base sequence represented by base numbers 3119 to 4317 of the base sequence represented by SEQ ID NO: 69 was read.
Further, a DNA that extends 5 ′ downstream from the base number 2838 of the base sequence represented by SEQ ID NO: 69 was cloned. Specifically, 3 μl (253 ng) of the total RNA prepared in Example 10 (3), 1.0 μl of CDS-primer attached to the kit and 1.0 μl of SMART II A oligo were mixed at 70 ° C. After incubating for 2 minutes, the mixture was kept on ice for 2 minutes. Using the reaction solution, 5 ′ RACE ready cDNA was prepared in the same manner as 3 ′ RACE ready cDNA preparation. Using this 5 ′ RACE ready cDNA as a template, PCR was performed to amplify the 5 ′ upstream region using KOD-plus- (TOYOBO). The PCR reaction solution is 2.5 μl of 5 ′ RACE ready cDNA, 5.0 μl of 10 × buffer, 5.0 μl of 2 mM dNTPs, 2.0 μl of 25 mM MgSO4, and 1.0 μl of KOD-Plus-, and the primers are attached to the kit. In addition, 5.0 μl of 10 × Universal Primer A Mix and 1.0 μl of 10 μM oligonucleotide solution consisting of the base sequence represented by SEQ ID NO: 71 were added, and the total volume was adjusted to 50 μl using sterilized distilled water. This reaction solution was kept at 94 ° C. for 2 minutes, and further, this was repeated 35 times with one cycle of holding at 94 ° C. for 15 seconds and then at 68 ° C. for 6 minutes. 1 μl of the PCR reaction solution thus obtained was used as a template, 5.0 μl of 10 × buffer, 5.0 μl of 2 mM dNTPs, 2.0 μl of 25 mM MgSO4 and 1.0 μl of KOD-Plus− were mixed, and 10 μM attached to the kit as a primer. Nested universal primer (1.0 μl) and a 10 μM oligonucleotide solution consisting of the base sequence represented by SEQ ID NO: 72 were added at 1.0 μl, and the total volume was adjusted to 50 μl using sterilized distilled water. This reaction solution was kept at 94 ° C. for 2 minutes, and further, this was repeated for 20 cycles with 94 ° C. for 15 seconds and then 68 ° C. for 6 minutes as one cycle. The PCR reaction solution and the pCR2.1-TOPO cloning vector (Invitrogen) were ligated according to the instruction manual attached to the cloning vector, and then introduced into E. coli JM109 (TaKaRa). Plasmid DNA was purified from the obtained Escherichia coli transformant using QIAprep Spin Miniprep Kit (QIAGEN). The obtained plasmid DNA was used as a template, and the nucleotide sequence was analyzed according to the method described in Example 11 (1) using primers having the nucleotide sequences represented by SEQ ID NOs: 28, 29, 77 to 82. As a result, the base sequence represented by base numbers 1 to 3042 of the base sequence represented by SEQ ID NO: 69 was read.
As a result of linking all the analyzed base sequences, the base sequence represented by SEQ ID NO: 69 was obtained. The base sequence represented by SEQ ID NO: 69 consisted of 4317 bases (including a stop codon) and was a base sequence encoding 1438 amino acid residues (SEQ ID NO: 68). The molecular weight of the protein having the amino acid sequence represented by SEQ ID NO: 68 was calculated to be 155296 Da.

(3) Isolation of RsOS-1 full-length rice mold fungus RsOS-1 full-length gene RsOS-1 gene of rice mold blight fungus prepared in Example 10 (5) using the cDNA of rice mold blight fungus as a template Was amplified by PCR. By performing a PCR reaction according to the method described in Example 11 (3) using the oligonucleotide consisting of the base sequence shown by SEQ ID NO: 85 and the oligonucleotide consisting of the base sequence shown by SEQ ID NO: 86 as primers, the sequence A DNA having the base sequence indicated by No. 69 was amplified.
PCR reaction was carried out using KOD-Plus- (TOYOBO) at 94 ° C for 2 minutes, followed by incubation at 94 ° C for 15 seconds, then at 55 ° C for 30 seconds and then at 68 ° C for 6 minutes as one cycle. Amplification was repeated for 35 cycles. The PCR reaction solution (50 μl) was 2.5 μl of 5 ′ RACE ready cDNA, 5.0 μl of 10 × buffer, 5.0 μl of 2 mM dNTPs, 2.0 μl of 25 mM MgSO4, 1.0 μl of 10 μM oligonucleotide primer, and sterile distilled water. 32.5 μl and KOD-Plus- were prepared by adding 1.0 μl.
A part of the PCR reaction solution after the reaction was separated by 1% agarose gel electrophoresis, and it was confirmed that about 4 kb of RsOS-1 gene was amplified by staining with ethidium bromide.

Example 18 (Construction of an expression plasmid for rice mold blight fungus RsOS-1 gene and production of transformed budding yeast)
First, the rice sheath blight fungus RsOS-1 gene was subcloned into the pCR2.1-TOPO cloning vector (Invitrogen). The reaction solution containing RsOS-1 gene prepared in Example 17 (3) was purified using QIAquick PCR Purification Kit (QIAGEN) according to the manual attached to the kit. According to the method described in Example 11 (1), 3′A addition was performed on the purified DNA of about 4 kb. The plasmid pCRRsOS1 was constructed by ligating the 3′A-added DNA and the pCR2.1-TOPO cloning vector (Invitrogen) according to the instruction manual attached to the cloning vector. The nucleotide sequence of the obtained plasmid was analyzed according to the method described in Example 11 (1). As a primer, an oligonucleotide consisting of any one of the base sequences shown in SEQ ID NOs: 28, 29, 70 to 73, 75, 77, 78, 81 to 84 was used. As a result, the base sequence shown in SEQ ID NO: 69 was obtained, and it was confirmed that the plasmid pCRRsOS1 contains the RsOS-1 gene.
An expression plasmid was constructed by cloning the RsOS-1 gene containing the plasmid pCR RsOS1 thus prepared into the yeast-E. Coli shuttle vector p415ADH. Plasmid pCRRsOS1 was digested with restriction enzymes SpeI and HindIII, while shuttle vector p415ADH was also digested with restriction enzymes SpeI and HindIII. After separating these by 0.8% agarose gel electrophoresis, the RsOS-1 gene fragment digested with the restriction enzymes SpeI and HindIII and the gel part containing the shuttle vector p415ADH digested with the restriction enzymes SpeI and HindIII were excised. Using the QIAquick Gel Extraction Kit (QIAGEN), collect the RsOS-1 gene fragment digested with the restriction enzymes SpeI and HindIII and the shuttle vector digested with the restriction enzymes SpeI and HindIII from the gel according to the manual attached to the kit. did. Using Ligation Kit Ver.2 (TaKaRa), construct the expression plasmid pADHRsOS1 by inserting the RsOS-1 gene between SpeI and HindIII of the multicloning site of the shuttle vector according to the manual attached to the kit. did. The base sequence of the obtained expression plasmid was analyzed according to the method described in Example 11 (1). As a primer, an oligonucleotide consisting of any one of the base sequences shown in SEQ ID NOs: 87, 88, 70 to 73, 75, 77, 78, 81 to 84 was used. As a result, the base sequence shown in SEQ ID NO: 69 was obtained, and it was confirmed that the expression plasmid pADHRsOS1 possesses the RsOS-1 gene.
The prepared expression plasmid pADH RsOS1 was introduced into the budding yeast strains AH22 and TM182 according to the method described in Example 2. In the obtained transformed budding yeast, the auxotrophy of leucine disappears, and the transformed budding yeast AH22 strain (AH22-RsOS1) is selected on the Glu-Leu agar medium, and the transformed budding yeast strain TM182 (TM182 -RsOS1) was selected on Gal-Ura-Leu agar medium. The obtained TM182-RsOS1 was confirmed to grow even when transplanted to a Glu-Ura-Leu medium.

Example 19 (Antimicrobial susceptibility test of transformed budding yeast TM182-RsOS1)
The transformed budding yeast AH22-RsOS1 prepared in Example 18 was cultured with shaking in Glu-Leu medium at 30 ° C. for 18 hours. As a control, the AH22 strain was similarly cultured with shaking in Glu medium at 30 ° C. for 18 hours. The absorbance at 600 nm in each of the grown transformed budding yeast cell suspensions was measured, and a cell suspension diluted with sterilized distilled water was prepared so that the absorbance was 0.1. Furthermore, a bacterial suspension in which transformed budding yeast AH22-RsOS1 was diluted 50 times with Glu-Leu medium and a bacterial suspension in which AH22 strain was diluted 50 times with Glu medium were prepared.
A solution in which five compounds represented by chemical formulas (1) to (5) (compounds (1) to (5)) are each dissolved in dimethyl sulfoxide (DMSO) so as to have a concentration of 600 ppm, and chemical formula Prepare a solution in which the two types of compounds shown in (6) and (7) (compounds (6) and (7)) are each dissolved in dimethyl sulfoxide (DMSO) to a concentration of 60 ppm. Two microplates were prepared in which 1.0 μl of each compound DMSO solution and DMSO as a control were dispensed in two locations (ie, two wells). On one of them, 100 μl each of the suspension of transformed budding yeast AH22-RsOS1 diluted and prepared as described above was dispensed and statically cultured at 30 ° C. for 29.8 hours. On the other sheet, 100 μl each of the suspension of the control yeast strain AH22 diluted and prepared as described above was dispensed and statically cultured at 30 ° C. for 24.8 hours. After incubation, the absorbance at 600 nm in each hole was measured with a microplate reader.
Similarly, the transformed budding yeast TM182-RsOS1 prepared in Example 18 was cultured in a Glu-Ura-Leu medium at 30 ° C. for 18 hours. The absorbance at 600 nm of the bacterial suspension of the grown transformed budding yeast was measured, and a bacterial suspension diluted with sterilized distilled water was prepared so that the absorbance was 0.1. Further, the transformed budding yeast TM182- A bacterial suspension in which RsOS1 was diluted 50 times with Glu-Ura-Leu medium was prepared. As a control, transformed budding yeast TM182-RsOS1 was cultured in Gal-Ura-Leu medium at 30 ° C. for 18 hours. The absorbance at 600 nm of the bacterial suspension of the grown transformed budding yeast was measured, and a bacterial suspension diluted with sterilized distilled water was prepared so that the absorbance was 0.1. Further, the transformed budding yeast TM182- A bacterial suspension in which RsOS1 was diluted 50-fold with Gal-Ura-Leu medium was prepared.
A solution in which five kinds of compounds represented by chemical formulas (1) to (5) (compounds (1) to (5)) are dissolved in dimethyl sulfoxide (DMSO) to a concentration of 600 ppm, respectively, Prepare a solution in which the two compounds shown in 6) and (7) (compounds (6) and (7)) are each dissolved in dimethyl sulfoxide (DMSO) so as to have a concentration of 60 ppm. Two microplates were prepared in which DMSO solution and DMSO as a control were dispensed at two locations (ie, 2 wells), 1.0 μl in each well. On one of them, 100 μl each of the suspension of transformed budding yeast TM182-RsOS1 diluted in Glu-Ura-Leu medium as described above was dispensed and incubated at 30 ° C. for 26.8 hours. . On the other side, as described above, 100 μl each of the suspension of transformed budding yeast TM182-RsOS1 diluted in Gal-Ura-Leu medium as a control was dispensed and cultured at 30 ° C. for 42.5 hours. did. After incubation, the absorbance at 600 nm in each hole was measured with a microplate reader.
Table 7 shows the degree of growth of each transformed budding yeast for the compounds represented by chemical formulas (1) to (7) (compounds (1) to (7)). The degree of growth of the transformed budding yeast was expressed as a percentage relative to the absorbance at 600 nm when the compound concentration was 0 ppm as 100. The degree of growth inhibition by each test substance of transformed budding yeast TM182-RsOS1 is larger than the degree of growth inhibition by each test substance of transformed budding yeast AH22-RsOS1, and transformed budding yeast TM182-RsOS1 -It was confirmed that the transformed cells had enhanced sensitivity to antibacterial active substances compared to RsOS1.

Example 20 (Isolation of an osmosensitive histidine kinase PiOS-1 gene having no transmembrane domain of tomato plague fungus)
(1) Analysis of Tomato Blight Fungus PiOS-1 Gene Fragment A PCR reaction solution, which was amplified in Example 10 (5) and performed using primer pair 6 with the rice mold blight fungus cDNA as a template, was used as QIAquick. Purification was performed using PCR Purification Kit (QIAGEN) according to the instructions attached to the kit. The amplified DNA obtained by purification was subjected to 3′A addition according to the method described in Example 11 (1). The 3′A-added DNA and the pCR2.1-TOPO cloning vector (Invitrogen) were ligated according to the instruction manual attached to the cloning vector, and then introduced into E. coli JM109 (TaKaRa).
A DNA fragment was amplified from the obtained Escherichia coli transformant by colony PCR using Ex Taq HS (TaKaRa). The PCR reaction solution (15 μl) is composed of 1.5 μl of 10 × buffer, 2.25 μl of 10 mM dNTPs, 0.15 μl of Ex Taq HS, 10 μM oligonucleotide solution consisting of the base sequence represented by SEQ ID NO: 28 and the base represented by SEQ ID NO: 29 0.4 μl each of 10 μM solution of oligonucleotide consisting of sequence and 10.3 μl of sterilized distilled water were mixed and prepared by adding a part of E. coli transformant colony. This reaction solution was kept at 97 ° C. for 2 minutes, and then kept at 97 ° C. for 15 seconds, then at 55 ° C. for 15 seconds and further at 72 ° C. for 3 minutes. The incubated PCR reaction solution was purified using a QIAquick PCR purification Kit (QIAGEN) according to the instruction manual attached to the kit. The nucleotide sequence was analyzed according to the method described in Example 11 (1) using the PCR amplified fragment obtained by purification as a template and using the oligonucleotide consisting of the nucleotide sequences represented by SEQ ID NOs: 28 and 29 as primers. As a result, the base sequence represented by SEQ ID NO: 89 containing the sequence of primer pair 6 was read.

(2) Analysis of full-length OS-1 homologous gene of tomato blight fungus OS-1
Using the SMART RACE cDNA Amplification Kit (CLONTECH), DNA extending to the 5 ′ upstream region of the base sequence represented by SEQ ID NO: 89 can be cloned according to the instruction manual attached to the kit. Specifically, 3 μl (200 ng) of the total RNA prepared in Example 10 (4) was mixed with 1.0 μl of CDS-primer attached to the kit and 1.0 μl of SMART IIA Oligo to prepare a reaction solution. Incubate at 70 ° C for 2 minutes, then keep on ice for 2 minutes. 2 μl of 5 × First-Strand buffer attached to the kit, 1 μl of 20 mM DTT, 1 μl of 10 mM dNTP Mix and 1 μl of PowerScript Reverse Transcriptase attached to the kit are added to the reaction mixture, and the mixture is incubated at 42 ° C. for 1.5 hours. 100 μl of Tricine-EDTA buffer attached to the kit is added to the reaction solution after incubation, and then incubated at 72 ° C. for 7 minutes to prepare 5 ′ RACE ready cDNA. Using this 5 ′ RACE ready cDNA as a template, PCR was performed to amplify the 5 ′ upstream region using KOD-plus- (TOYOBO). The PCR reaction solution was mixed with 2.5 μl of 5 ′ RACE ready cDNA, 5.0 μl of 10 × buffer, 5.0 μl of 2 mM dNTPs, 2.0 μl of 25 mM MgSO4, 1.0 μl of KOD-Plus-, and a primer was attached to the kit. Add 5.0 μl of 10x Universal Primer A Mix and 1.0 μl of a 10 μM oligonucleotide solution consisting of 20-30 bases selected from the complementary sequence of SEQ ID NO: 89, and use sterilized distilled water to 50 μl. This reaction solution is kept at 94 ° C. for 2 minutes, and this is further repeated 35 times with one cycle of holding at 94 ° C. for 15 seconds and then at 68 ° C. for 5 minutes. The PCR reaction solution is purified using QIAquick PCR Purification Kit (QIAGEN) according to the instruction manual attached to the kit, and then the purified DNA is purified according to the method described in Example 11 (1). Add 3'A. The 3'A-added DNA and the pCR2.1-TOPO cloning vector (Invitrogen) are ligated according to the instruction manual attached to the cloning vector, and then introduced into E. coli JM109 (TaKaRa). Plasmid DNA is purified from the resulting E. coli transformant using QIAprep Spin Miniprep Kit (QIAGEN). The obtained plasmid DNA is used as a template, and the nucleotide sequence is analyzed according to the method described in Example 11 (1) using a primer consisting of the nucleotide sequence represented by SEQ ID NOs: 28, 29 and the like. As a result, the nucleotide sequence of a part of the OS-1 homologous gene of the tomato plague fungus (including the translation initiation codon of the osmotic sensitive histidine kinase PiOS-1 gene having no transmembrane region of the tomato plague fungus 5) (Base sequence of end region) can be read.
Furthermore, DNA extending to the 3 ′ downstream region of the base sequence represented by SEQ ID NO: 89 can be cloned. Specifically, 3 μl (200 ng) of the total RNA prepared in Example 10 (4) was mixed with 1.0 μl of CDS-primer attached to the kit and 1.0 μl of sterilized distilled water. Incubate for 2 minutes, then keep on ice for 2 minutes. Using the reaction solution, 3 ′ RACE ready cDNA is prepared in the same manner as 5 ′ RACE ready cDNA preparation. Using this 3 ′ RACE ready cDNA as a template, PCR is performed to amplify the 3 ′ downstream region. The PCR reaction solution was prepared by mixing 2.5 μl of 5 'RACE ready cDNA, 5.0 μl of 10x Advantage 2 buffer, 1.0 μl of 10 mM dNTP Mix and 1.0 μl of 50x Advantage 2 Polymerase Mix attached to the kit as primers. Add 5.0 μl of 10 × Universal Primer A Mix attached to the kit and 1.0 μl of 10 μM oligonucleotide solution consisting of 20-30 bases selected from the base sequence shown in SEQ ID NO: 89, and use sterile distilled water The total volume is 50 μl. This reaction solution was incubated for 5 cycles at 94 ° C for 5 seconds and then at 72 ° C for 4 minutes, and this was repeated 5 cycles. This is repeated 5 times as a cycle, and further, this is repeated 25 cycles with 94 ° C. for 5 seconds, then 68 ° C. for 10 seconds, then 72 ° C. for 4 minutes, and then kept at 72 ° C. for 7 minutes. The PCR reaction solution and the pCR2.1-TOPO cloning vector (Invitrogen) are ligated according to the instruction manual attached to the cloning vector, and then introduced into E. coli JM109 (TaKaRa). Plasmid DNA is purified from the resulting E. coli transformant using QIAprep Spin Miniprep Kit (QIAGEN). Using the obtained plasmid DNA as a template, the base sequence is analyzed according to the method described in Example 11 (1) using a primer consisting of the base sequence represented by SEQ ID NOs: 28, 29 and the like. As a result, the nucleotide sequence of a part of the OS-1 homologous gene of the tomato plague fungus (including the translational stop codon of the osmotic sensitive histidine kinase PiOS-1 gene that does not have the transmembrane region of the tomato plague fungus 3 (Base sequence of end region) can be read.
As a result of linking all the analyzed base sequences, the entire base sequence of the OS-1 homologous gene of tomato blight fungus containing SEQ ID NO: 89 can be clarified.

(3) Isolation of Tomato Blight Fungus OS-1 Homologous Gene Full-length Gene Using the cDNA prepared in Example 10 (4) as a template, the tomato blight fungus OS-1 homologous gene can be amplified by PCR. . 5 ′ of an oligonucleotide having a base sequence from the 5 ′ end to the 20th base including the start codon of the base sequence of the OS-1 homologous gene of tomato plague fungus, which was clarified by the method of Example 20 (2) From the 3 ′ end side containing the end codon of the base sequence of the OS-1 homologous gene of the tomato plague fungus, which was clarified by the method of Example 20 (2), and the oligonucleotide to which the ACGACAGT sequence was added on the terminal side According to the method described in Example 11 (3), using as an primer an oligonucleotide having a sequence complementary to the sequence in which the sequence of AAGCTTCAG is added to the 3 ′ end of the oligonucleotide consisting of the sequence up to the 20th base By performing a PCR reaction, a DNA containing the OS-1 homologous gene of tomato blight fungus and having a restriction enzyme SpeI recognition sequence immediately before the start codon and a restriction enzyme HindIII recognition sequence immediately after the stop codon is obtained. To width. A part of the PCR reaction solution after the reaction is separated by 1% agarose gel electrophoresis, and ethidium bromide staining is performed to confirm that the OS-1 homologous gene of about 4 kb tomato plague fungus is amplified.

Example 21 (Construction of expression plasmid of tomato plague fungus OS-1 homologous gene and production of transformed budding yeast)
First, the tomato blight fungus OS-1 homologous gene is subcloned into the pCR2.1-TOPO cloning vector (Invitrogen). The reaction solution containing the OS-1 homologous gene of tomato blight fungus prepared in Example 20 (3) is purified using QIAquick PCR Purification Kit (QIAGEN) according to the manual attached to the kit. 3′A addition is performed on the approximately 4 kb DNA obtained by purification according to the method described in Example 11 (1). The plasmid pCRPiOS1 is constructed by ligating the 3′A added DNA of about 4 kb and the pCR2.1-TOPO cloning vector (Invitrogen) according to the instruction manual attached to the cloning vector. The nucleotide sequence of the obtained plasmid was analyzed according to the method described in Example 11 (1). As a primer, an oligonucleotide having a base sequence represented by SEQ ID NO: 28, 29 or the like is used. As a result, it can be confirmed that the plasmid pCRPiOS1 possesses a tomato plague fungus OS-1 homologous gene containing SEQ ID NO: 89.
An expression plasmid is constructed by cloning the tomato blight fungus OS-1 homologous gene contained in the thus prepared plasmid pCR PiOS1 into the yeast-E. Coli shuttle vector p415ADH. Plasmid pCRPiOS1 is digested with restriction enzymes SpeI and HindIII, while shuttle vector p415ADH is also digested with restriction enzymes SpeI and HindIII. The gel part containing the shuttle vector p415ADH digested with restriction enzymes SpeI and HindIII and the tomato plague fungus OS-1 homologous gene fragment digested with restriction enzymes SpeI and HindIII after separation by 0.8% agarose gel electrophoresis, respectively. Using the QIAquick Gel Extraction Kit (QIAGEN), the StOS-1 gene fragment digested with the restriction enzymes SpeI and HindIII and the restriction enzymes SpeI and HindIII were digested from the gel according to the manual attached to the kit. Collect the shuttle vector. Using Ligation Kit Ver.2 (TaKaRa), construct the expression plasmid pADHPiOS1 by inserting the StOS-1 gene between SpeI and HindIII of the multicloning site of the shuttle vector according to the manual attached to the kit To do. The base sequence of the obtained expression plasmid is analyzed according to the method described in Example 11 (1). As a primer, an oligonucleotide having a base sequence represented by SEQ ID NO: 28, 29 or the like is used. As a result, the base sequence of the OS-1 homologous gene of tomato blight fungus containing SEQ ID NO: 89 is obtained, and it can be confirmed that the expression plasmid pADHPiOS1 has the tomato blight fungus OS-1 homologous gene.
The prepared expression plasmid pADH PiOS1 is introduced into the budding yeast strains AH22 and TM182 according to the method described in Example 2. In the resulting transformed budding yeast, the auxotrophy of leucine disappears, and the transformed budding yeast AH22 strain (AH22-PiOS1) is selected on a Glu-Leu agar medium, and the transformed budding yeast strain TM182 (TM182 -Select PiOS1) on Gal-Ura-Leu agar medium. The obtained TM182-PiOS1 is confirmed to grow even when transplanted to Glu-Ura-Leu medium.

Example 22 (Antimicrobial susceptibility test of transformed budding yeast TM182-PiOS1)
The transformed budding yeast AH22-PiOS1 prepared in Example 21 is cultured with shaking at 30 ° C. in a Glu-Leu medium. As a control, the AH22 strain is similarly cultured with shaking in Glu medium at 30 ° C. The absorbance at 600 nm in each of the grown transformed budding yeast cell suspensions is measured, and a cell suspension diluted with sterile distilled water is prepared so that the absorbance is 0.1. Furthermore, a bacterial suspension in which transformed budding yeast AH22-PiOS1 is diluted 50 times with Glu-Leu medium and a bacterial suspension in which AH22 strain is diluted 50 times with Glu medium are prepared.
A solution in which seven kinds of compounds represented by chemical formulas (1) to (7) (compounds (1) to (7)) are dissolved in dimethyl sulfoxide (DMSO) is prepared, and DMSO solution as a control and DMSO as a control. Two microplates are prepared by dispensing 1.0 μl in each hole in two locations (ie, two wells). On one of them, 100 μl each of the suspension of transformed budding yeast AH22-PiOS1 diluted and prepared as described above is dispensed and statically cultured at 30 ° C. On the other sheet, 100 μl of the suspension of the control yeast AH22 strain diluted as described above is dispensed and statically cultured at 30 ° C. After incubation, the absorbance at 600 nm in each well is measured with a microplate reader.
Similarly, the transformed budding yeast TM182-PiOS1 prepared in Example 21 is cultured at 30 ° C. in a Glu-Ura-Leu medium. The absorbance at 600 nm of the bacterial suspension of the grown transformed budding yeast is measured, and a bacterial suspension diluted with sterile distilled water is prepared so that the absorbance is 0.1. Furthermore, a bacterial suspension in which transformed budding yeast TM182-PiOS1 was diluted 50 times with Glu-Ura-Leu medium and a bacterial suspension diluted 50 times with Gal-Ura-Leu medium as a control. Prepare.
A solution in which seven kinds of compounds represented by chemical formulas (1) to (7) (compounds (1) to (7)) are dissolved in dimethyl sulfoxide (DMSO) is prepared, and DMSO solution as a control and DMSO as a control. Two microplates are prepared by dispensing 1.0 μl in each hole in two locations (ie, two wells). On one of them, 100 μl of the suspension of the transformed budding yeast TM182-PiOS1 diluted with Glu-Ura-Leu medium as described above is dispensed and statically cultured at 30 ° C. On the other sheet, as described above, 100 μl of the suspension of transformed budding yeast TM182-PiOS1 diluted with Gal-Ura-Leu medium as a control is dispensed and statically cultured at 30 ° C. After incubation, the absorbance at 600 nm in each well is measured with a microplate reader.
The degree of growth inhibition by each test substance of transformed budding yeast TM182-PiOS1 is greater than the degree of growth inhibition by each test substance of transformed budding yeast AH22-PiOS1, and transformed budding yeast TM182-PiOS1 is transformed with transformed budding yeast AH22 -It can be confirmed that the transformed cells have enhanced sensitivity to antibacterial active substances compared to PiOS1.

The composition of the medium used in the present invention is described below.
(A) Glu medium
Bacto-yeast nitrogen base without amino acids 6.7g, Glucose 20g, Drop-out mix (1) 2.0g, Distilled water 1000ml (b) Glu-Leu medium
Bacto-yeast nitrogen base without amino acids 6.7g, Glucose 20g, Drop-out mix (2) 2.0g, Distilled water 1000ml
(C) Glu-Ura-Leu medium
Bacto-yeast nitrogen base without amino acids 6.7g, Glucose 20g, Drop-out mix (3) 2.0g,
Distilled water 1000ml
(D) Gal-Ura-Leu medium
Bacto-yeast nitrogen base without amino acids 6.7g,
Galactose 20g, Drop-out mix (3) 2.0g,
Distilled water 1000ml

Drop-out mix (1):
Adenine 0.5g, Lysine 2.0g, Alanine 2.0g, Methionine 2.0g, Arginine 2.0g, para-Aminobenzoic acid 0.2g, Asparagine 2.0g, Phenylalanine 2.0g, Aspartic acid 2.0g, Proline 2.0g, Cysteine 2.0g, Serine 2.0 g, Glutamine 2.0g, Threonine 2.0g, Glutamic acid 2.0g, Tryptophan 2.0g, Glycine 2.0g, Tyrosine 2.0g, Histidine 2.0g, Valine 2.0g, Inositol 2.0g, Isoleucine 2.0g, Uracil 2.0g, Leucine 10.0g , Distilled water 1000ml
Drop-out mix (2): Drop-out mix (1) excluding Leucine (10.0g)
Drop-out mix (3): Drop-out mix (1) excluding Uracil (2.0g) and Leucine (10.0g)

(E) Glu agar medium A solid medium in which 2% (W / V) agar is added to the medium (a).
(F) Glu-Leu agar medium A solid medium in which 2% (W / V) agar is added to the medium (b).
(G) Glu-Ura-Leu agar medium A solid medium in which 2% (W / V) agar is added to the medium (c).
(H) Gal-Ura-Leu agar medium A solid medium in which 2% (W / V) agar is added to the medium (d).

  According to the present invention, it is possible to provide a transformed cell with enhanced sensitivity to an antibacterial active substance, and furthermore, an antibacterial activity test method for a test substance using the transformed cell, a method for searching for an antibacterial active substance using the method, etc. Was made available.

SEQ ID NO: 3
Oligonucleotide primer designed for PCR SEQ ID NO: 4
Oligonucleotide primer designed for PCR SEQ ID NO: 5
Oligonucleotide primer designed for sequencing SEQ ID NO: 6
Oligonucleotide primer designed for sequencing SEQ ID NO: 7
Oligonucleotide primer designed for sequencing SEQ ID NO: 8
Oligonucleotide primer designed for sequencing SEQ ID NO: 9
Oligonucleotide primer designed for sequencing SEQ ID NO: 10
Oligonucleotide primer designed for sequencing SEQ ID NO: 11
Oligonucleotide primer designed for sequencing SEQ ID NO: 12
Oligonucleotide primer designed for sequencing SEQ ID NO: 15
Oligonucleotide primer designed for PCR SEQ ID NO: 18
Oligonucleotide primer designed for PCR SEQ ID NO: 19
Oligonucleotide primer designed for PCR SEQ ID NO: 20
Oligonucleotide primer designed for sequencing SEQ ID NO: 21
Oligonucleotide primer designed for sequencing SEQ ID NO: 22
Oligonucleotide primer designed for sequencing SEQ ID NO: 23
Oligonucleotide primer designed for sequencing SEQ ID NO: 24
Oligonucleotide primer designed for sequencing SEQ ID NO: 25
Oligonucleotide primer designed for sequencing SEQ ID NO: 26
Oligonucleotide primer designed for sequencing SEQ ID NO: 27
Oligonucleotide primer designed for sequencing SEQ ID NO: 28
Oligonucleotide primer designed for sequencing SEQ ID NO: 29
Oligonucleotide primer designed for sequencing SEQ ID NO: 30
Oligonucleotide primer designed for sequencing SEQ ID NO: 31
Oligonucleotide primer designed for PCR SEQ ID NO: 32
Oligonucleotide primer designed for PCR SEQ ID NO: 33
Oligonucleotide primer designed for PCR SEQ ID NO: 34
Oligonucleotide primer designed for PCR SEQ ID NO: 35
Oligonucleotide primer designed for PCR SEQ ID NO: 36
Oligonucleotide primer designed for PCR SEQ ID NO: 37
Oligonucleotide primer designed for PCR SEQ ID NO: 38
Oligonucleotide primer designed for PCR SEQ ID NO: 39
Oligonucleotide primer designed for PCR SEQ ID NO: 40
Oligonucleotide primer designed for PCR SEQ ID NO: 43
Oligonucleotide primer designed for PCR SEQ ID NO: 44
Oligonucleotide primer designed for PCR SEQ ID NO: 45
Oligonucleotide primer designed for sequencing SEQ ID NO: 46
Oligonucleotide primer designed for sequencing SEQ ID NO: 47
Oligonucleotide primer designed for sequencing SEQ ID NO: 48
Oligonucleotide primer designed for sequencing SEQ ID NO: 49
Oligonucleotide primer designed for sequencing SEQ ID NO: 50
Oligonucleotide primer designed for sequencing SEQ ID NO: 51
Oligonucleotide primer designed for sequencing SEQ ID NO: 52
Oligonucleotide primer designed for PCR SEQ ID NO: 53
Oligonucleotide primer designed for PCR SEQ ID NO: 54
Oligonucleotide primer designed for PCR SEQ ID NO: 57
Oligonucleotide primer designed for PCR SEQ ID NO: 58
Oligonucleotide primer designed for PCR SEQ ID NO: 59
Oligonucleotide primer designed for sequencing SEQ ID NO: 60
Oligonucleotide primer designed for sequencing SEQ ID NO: 61
Oligonucleotide primer designed for sequencing SEQ ID NO: 62
Oligonucleotide primer designed for sequencing SEQ ID NO: 63
Oligonucleotide primer designed for sequencing SEQ ID NO: 64
Oligonucleotide primer designed for PCR SEQ ID NO: 65
Oligonucleotide primer designed for PCR SEQ ID NO: 66
Oligonucleotide primer designed for sequencing SEQ ID NO: 67
Oligonucleotide primer designed for sequencing SEQ ID NO: 70
Oligonucleotide primer designed for PCR SEQ ID NO: 71
Oligonucleotide primer designed for PCR SEQ ID NO: 72
Oligonucleotide primer designed for PCR SEQ ID NO: 73
Oligonucleotide primer designed for PCR SEQ ID NO: 74
Oligonucleotide primer designed for PCR SEQ ID NO: 75
Oligonucleotide primer designed for PCR SEQ ID NO: 76
Oligonucleotide primer designed for PCR SEQ ID NO: 77
Oligonucleotide primer designed for PCR SEQ ID NO: 78
Oligonucleotide primer designed for PCR SEQ ID NO: 79
Oligonucleotide primer designed for PCR SEQ ID NO: 80
Oligonucleotide primer designed for PCR SEQ ID NO: 81
Oligonucleotide primer designed for PCR SEQ ID NO: 82
Oligonucleotide primer designed for PCR SEQ ID NO: 83
Oligonucleotide primer designed for PCR SEQ ID NO: 84
Oligonucleotide primer designed for PCR SEQ ID NO: 85
Oligonucleotide primer designed for PCR SEQ ID NO: 86
Oligonucleotide primer designed for PCR SEQ ID NO: 87
Oligonucleotide primer designed for PCR SEQ ID NO: 88
Oligonucleotide primers designed for PCR

Claims (11)

A cell deficient in at least one hybrid sensor kinase is composed of a base sequence encoding the amino acid sequence of an osmotic pressure sensitive histidine kinase that does not have a transmembrane region, and complements the function of the hybrid sensor kinase deficient in the cell. A transformed cell, wherein a gene is introduced in a functional form. 2. The transformed cell according to claim 1, wherein the cell is a microorganism. The transformed cell according to claim 1, wherein the microorganism is a budding yeast. The gene comprising the base sequence encoding the amino acid sequence of osmotic pressure sensitive histidine kinase that does not have a transmembrane region is any of dicarboximide antibacterial active substance, aromatic hydrocarbon antibacterial active substance, and phenylpyrrole antibacterial active substance. 2. A gene comprising a nucleotide sequence encoding an amino acid sequence of histidine kinase which is a histidine kinase having a mutation resistant to osmotic pressure and does not have a transmembrane region. Transformed cells. 2. The transformed cell according to claim 1, wherein the osmosensitive histidine kinase not having a transmembrane region is an osmotic sensitivity histidine kinase having no transmembrane region derived from a phytopathogenic filamentous fungus. Osmotic-sensitive histidine kinase that does not have a transmembrane region is derived from gray mold fungi, rice blast fungus, spinach wilt fungus, wheat leaf blight fungus, rice blight fungus or tomato blight fungus The transformed cell according to claim 1, which is an osmotic pressure sensitive histidine kinase having no transmembrane region. The amino acid sequence of an osmotic pressure sensitive histidine kinase having no transmembrane region is an amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 16, SEQ ID NO: 41, SEQ ID NO: 55 or SEQ ID NO: 68. The transformed cell according to 1. The base sequence encoding the amino acid sequence of an osmotic pressure sensitive histidine kinase that does not have a transmembrane region is a base sequence represented by SEQ ID NO: 2, SEQ ID NO: 17, SEQ ID NO: 42, SEQ ID NO: 56 or SEQ ID NO: 69. The transformed cell according to claim 1, wherein A method for assaying antibacterial activity of a substance,
A first step of culturing the transformed cell according to claim 1 in the presence of a test substance;
A second step of measuring an intracellular signal transduction amount from an osmotic pressure sensitive histidine kinase not having a transmembrane region expressed in a transformed cell cultured in the first step or an index value correlated therewith,
A third step of evaluating the antibacterial activity of the test substance based on the difference between the intracellular signal transduction amount measured in the second step or an index value correlated therewith and a control,
A test method characterized by comprising: The assay method according to claim 9, wherein the intracellular signal transduction amount from an osmotic pressure sensitive histidine kinase not having a transmembrane region or an index value correlated therewith is the growth amount of the transformed cell. . An antibacterial active substance search method, comprising selecting an antibacterial active substance based on the antibacterial activity evaluated by the assay method according to claim 9.