JP4817751B2 - Novel antibacterial proteins and their uses - Google Patents

Description

  The present invention relates to a novel antibacterial cystatin protein and a nucleic acid encoding the same. The present invention also relates to a plant having resistance to pathogenic bacteria (particularly snow rot fungus) or abiotic stress, and a method for producing the same.

  Cysteine protease inhibitors were first identified in mammalian tissues in the late 50s, and papain-like cysteine peptidase inhibitors isolated from chicken egg white have been named “cystatin” (Non-Patent Document 1). Plant cystatins were first reported in rice (Non-Patent Document 2), after which phytocystatins have been identified in many monocotyledonous and dicotyledonous plants. Recently, plant cystatins have been classified into distinct subfamilies (Non-Patent Document 3). Most phytocystatins have a molecular weight of 12-16 kDa, show significant amino acid sequence homology, and do not contain disulfide bonds. Cysteine protease inhibitors are well characterized in rice (such as Non-Patent Document 2), corn (such as Non-Patent Document 4), and soybean (such as Non-Patent Document 5). Although phytocystatin is mainly purified from seeds (Non-patent Document 6 and the like), it is also detected in tubers, leaves, fruits, pollen, flower buds and petals (Non-Patent Document 7 and the like). Cystatin expression and stress response results have been reported during specific plant development periods. Involvement of cysteine protease inhibitors in protein metabolism during seed maturation and germination has also been reported (Non-patent Document 8, etc.). The protective function of cystatin against pests and pathogens has been proved by transgenic plants overexpressing cysteine protease inhibitors (Non-patent Document 9, etc.).

  On the other hand, winter cereals and perennial pastures survive even in winter when subzero temperatures and pathogens attack. The cold tolerance of plants has increased substantially after a period of exposure to low but non-freezing temperatures. In the northern region, winter cereals, including wheat, barley, rye and many pastures, are often attacked by the psychrophilic fungi, M. nivale, Thphula spp., Sclerotinia borealis, Protected against covered freezing. In wintering perennial pastures that are resistant to low temperatures, acquisition of low-temperature tolerance is correlated with acquisition of snow rot fungus resistance (Non-patent Document 10). Snow rot infections are opportunistic and are unique in that they gradually progress over time under snow. Therefore, the mechanism of snow rot resistance is hardly elucidated in molecular physiology.

  As a countermeasure against snow rot fungi, chemical treatment before root snow is performed. Iminocudazine acetate, thiophanate methyl, organic copper, copper sulfate, etc. are used, and certain effects are seen. However, it is difficult to determine the appropriate time for spraying with chemicals, and if spraying is early, runoff to the soil due to rainfall occurs and the effect decreases. In addition, a great load on the environment is also a problem. Another problem is that the fluctuation of the root snow period is large every year and the effect is not stable. Moreover, it is a cost burden to a farmer that the chemical | medical agent used for every pathogenic microbe differs.

  In addition, as another method of plant defense against microorganisms, many reports have been made of introducing antibacterial protein genes into plants to improve resistance to pathogenic bacteria (Non-patent Documents 11 and 12, etc.). However, the effect differs depending on the type of pathogenic bacteria, and there has been no example of improving the resistance to snow rot fungi, which has become a great wonder in the production of wintering crops such as winter wheat in cold regions.

Therefore, methods and means for imparting resistance to snow rot fungi to plants have been desired.
Barret, AJ, Trends Biochem. Sci. 12, 193-196, 1987 Abe, K. et al., J. Biol. Chem. 262, 16793-16797, 1987 Margis, E. et al., Arch. Biochem. Biophys. 359, 24-30, 1998 Abe, M. et al., Eur. J. Biochem. 209, 933-937, 1992 Hines, ME et al., J. Agric. Food Chem. 39, 1515-1520, 1991 Hirashiki, I. et al., J. Biochem (Tokyo) 108, 2259-2264, 1990 Diop, NN et al., FEBS Lett. 577, 545-550, 2004 Salmia, MA et al., Physiol. Plant 48, 266-270, 1980 Inanaga, H. et al., Biosci. Biotechnol. Biochem. 65, 2259-2264, 2001 Tronsmo, AM et al., Acta. Agric. Scand. 43, 89-96, 1993 Cocca et al., Plant Mol Biol. 54, 245, 2004 Clausen et al., Nat Biotechnol. Volume 18, 446, 2000

  Then, in view of the actual condition mentioned above, an object of this invention is to provide the method and means for producing the plant which shows the resistance with respect to a snow rot fungus.

  As a result of intensive studies to solve the above problems, the present inventor succeeded in isolating a cDNA encoding a new winter wheat-derived cystatin having a structure different from that of a known cereal cystatin. The present invention was found to impart resistance to snow rot fungi and abiotic stress.

That is, the present invention includes the following.
1. The following protein (a) or (b).
(A) a protein comprising the amino acid sequence 38 to 243 of the amino acid sequence shown in SEQ ID NO: 2 (b) one or several amino acids are substituted in the amino acid sequence 38 to 243 of the amino acid sequence shown in SEQ ID NO: 2 1. a protein comprising an amino acid sequence deleted, inserted or added and having antibacterial activity A nucleic acid encoding the following protein (a) or (b).
(A) a protein comprising the amino acid sequence 38 to 243 of the amino acid sequence shown in SEQ ID NO: 2 (b) one or several amino acids are substituted in the amino acid sequence 38 to 243 of the amino acid sequence shown in SEQ ID NO: 2 2. a protein comprising an amino acid sequence deleted, inserted or added and having antibacterial activity; The following nucleic acid (a) or (b):
(A) a nucleic acid consisting of the 166th to 786th base sequence of the base sequence shown in SEQ ID NO: 1, and (b) a nucleic acid consisting of a sequence complementary to the 166th to 786th base sequence of the base sequence shown in SEQ ID NO: 1. Nucleic acid that hybridizes under stringent conditions and encodes a protein having antibacterial activity

4). The following polypeptide (a) or (b).
(A) a polypeptide comprising amino acids 40 to 129 of the amino acid sequence shown in SEQ ID NO: 2 (b) substitution of one or several amino acids in the amino acid sequence of 40 to 129 of the amino acid sequence shown in SEQ ID NO: 2 4. A polypeptide comprising an amino acid sequence deleted, inserted or added and having antibacterial activity A nucleic acid encoding the following polypeptide (a) or (b).
(A) a polypeptide comprising amino acids 40 to 129 of the amino acid sequence shown in SEQ ID NO: 2 (b) substitution of one or several amino acids in the amino acid sequence of 40 to 129 of the amino acid sequence shown in SEQ ID NO: 2 5. A polypeptide comprising an amino acid sequence deleted, inserted or added and having antibacterial activity The following nucleic acid (a) or (b):
(A) a nucleic acid consisting of the 172nd to 441st base sequences of the base sequence shown in SEQ ID NO: 1 (b) a nucleic acid consisting of a sequence complementary to the 172nd to 441st base sequences of the base sequence shown to SEQ ID NO: 1 Nucleic acid that hybridizes under stringent conditions and encodes a polypeptide having antibacterial activity

7). The following polypeptide (a) or (b).
(A) a polypeptide comprising amino acids 150 to 210 of the amino acid sequence shown in SEQ ID NO: 2 (b) substitution of one or several amino acids in the amino acid sequence 150 to 210 of the amino acid sequence shown in SEQ ID NO: 2 7. A polypeptide comprising an amino acid sequence deleted, inserted or added and having antibacterial activity. A nucleic acid encoding the following polypeptide (a) or (b).
(A) a polypeptide comprising amino acids 150 to 210 of the amino acid sequence shown in SEQ ID NO: 2 (b) substitution of one or several amino acids in the amino acid sequence 150 to 210 of the amino acid sequence shown in SEQ ID NO: 2 8. A polypeptide comprising an amino acid sequence deleted, inserted or added and having antibacterial activity. The following nucleic acid (a) or (b):
(A) a nucleic acid comprising the base sequences 502 to 684 of the base sequence represented by SEQ ID NO: 1, and (b) a nucleic acid comprising a sequence complementary to the base sequences 502 to 684 of the base sequence represented by SEQ ID NO: 1. Nucleic acid that hybridizes under stringent conditions and encodes a polypeptide having antibacterial activity

10. The following protein (a) or (b).
(A) a protein comprising the amino acid sequence shown in SEQ ID NO: 2 (b) consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence shown in SEQ ID NO: 2, and 10. Protein having antibacterial activity A nucleic acid encoding the following protein (a) or (b).
(A) a protein comprising the amino acid sequence shown in SEQ ID NO: 2 (b) consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence shown in SEQ ID NO: 2, and 11. Protein having antibacterial activity The following nucleic acid (a) or (b):
(A) Nucleic acid consisting of the base sequence shown in SEQ ID NO: 1 (b) Hybridizing with a nucleic acid consisting of a sequence complementary to the base sequence shown in SEQ ID NO: 1 under stringent conditions and having antibacterial activity Nucleic acids encoding proteins

13. A recombinant vector comprising the nucleic acid.
14 A transformant transformed with the nucleic acid or the recombinant vector.
The transformant can be any cell, microorganism, plant, animal, etc., but is preferably a plant. Moreover, as a plant, although not limited, a plant body, a plant organ, a plant tissue, a plant culture cell, etc. are included. The plant is preferably a plant belonging to the family Gramineae, particularly wheat.
Moreover, the said transformant shows resistance with respect to snow rot fungi, for example, a red snow rot fungus. Alternatively, the transformant may exhibit resistance to abiotic stress selected from the group consisting of low temperature, salt and drying.

15. Seeds obtained from the transformant.
16. An antibody against the protein or polypeptide.
17. A method for producing a cystatin protein, comprising culturing the transformant and collecting a protein from the obtained culture.

18. A method for producing a resistant plant, comprising a step of transforming a plant using the nucleic acid or the recombinant vector, and a step of regenerating the plant from the resulting transformant.
By the above method, a plant resistant to snow rot fungi is produced. Also, by the above method, a plant resistant to abiotic stress selected from the group consisting of low temperature, salt and drying is produced. Plants include, but are not limited to, plants belonging to the family Gramineae, preferably wheat.

19. An antibacterial agent comprising at least one of the following (a) to (h):
(A) the protein according to 1 or 10 above (b) the polypeptide according to 4 or 7 above (c) the nucleic acid according to 2 or 11 above (d) the nucleic acid according to 5 or 8 above (e) the nucleic acid according to 3 or 12 above Nucleic acid (f) Nucleic acid according to item 6 or 9 (g) Recombinant vector according to item 13 above (h) Transformant according to item 14 above The antibacterial agent is preferably used in plants. The antibacterial agent has antibacterial activity against snow rot fungi.

  The present invention provides a protein (polypeptide) that imparts antibacterial activity to various organisms including plants and a gene encoding the same. Moreover, by using the protein and a gene encoding the protein, a plant (Gramineae plant, particularly wheat) having improved resistance to pathogenic bacteria and abiotic stress can be produced. In addition, an antibacterial agent using the protein and a gene encoding the protein is also provided.

Hereinafter, the present invention will be described in detail.
1. TECHNICAL FIELD The present invention relates to a novel cystatin protein derived from wheat (TaMDC1) and a gene encoding the same. Cystatin is a general term for endogenous inhibitor proteins that specifically inhibit the cysteine protease group belonging to the papain family. The TaMDC1 protein consists of about 23 kDa, has a signal sequence at the N-terminus, and has a cystatin domain (domain I) that is homologous to plant cystatins represented by oryzastatin, and a cystatin-like domain (domain II) that exhibits low homology to plant cystatin 2) (shown in gray background in FIG. 1). Putative domain I (40-129, FIG. 1) showed 99.1% identity when aligned with the CY domain of other plant cystatins. Further, ^{Gly 43,} the reaction site motif ^{QXVXG (Q 86 T 87 V 88} A 89 G 90), W 117 motifs, and advanced as required to cysteine protease inhibitory activity, including LARFAV motifs like that are highly conserved in phyto cystatin All of the conserved motifs were included (shown in FIG. 1 with black background and white letters). The putative domain II of TaMDC1 (No. 150-210, FIG. 1), which forms the C-terminal extension, only partially matches the CY domain of other plant cystatins (57%) and is conserved above The motif was not found in this domain II.

  TaMDC1 protein has cystatin activity, that is, protease inhibitory activity (papain inhibition) and antibacterial activity. This antibacterial activity is such a strong activity that it almost completely inhibits the growth of cells at a low concentration of 50 μg / ml against Microdochium nivare, which is one type of filamentous fungus. Here, domain I of TaMDC1 protein exhibits both cystatin activity and antibacterial activity, and domain II does not exhibit cystatin activity but exhibits antibacterial activity. Therefore, the polypeptide containing domain II of TaMDC1 protein exhibits antibacterial activity without inhibiting protease.

  Moreover, in the Example mentioned later, it was confirmed that the expression of TaMDC1 protein is induced in the process of low-temperature acclimation, and is also induced under salt, drying and abscisic acid (ABA) treatment. Thus, TaMDC1 protein may be involved in resistance to abiotic stresses such as low temperature, salt, desiccation and abscisic acid.

(2) Cloning of gene In the present invention, the gene encoding TaMDC1 protein is screened from RT-PCR or cDNA library using mRNA purified from RNA extracted from plant tissue, so that wheat shoot, crown It can be obtained from tissue or leaf tissue. In particular, since expression of the TaMDC1 gene is induced in the process of cold acclimation, it is preferable to extract RNA from wheat shoots, crown tissues or leaf tissues that have been cold acclimated. However, the plant that is the source of the gene of the present invention is not limited to wheat shoots, crown tissues, and leaf tissues. For example, grown plants, plant organs (eg leaves, petals, stems, etc.), plants It may be a tissue (for example, epidermis, phloem, soft tissue, xylem, vascular bundle, fence-like tissue, spongy tissue, etc.) or plant cultured cells (eg, callus). Furthermore, the plant used as the source of the gene of the present invention is not particularly limited as long as it belongs to the genus Wheat (Triticum). For example, wheat (Triticum aestivum), particularly winter wheat is preferable.

  Extraction of mRNA from wheat and preparation of a cDNA library can be performed according to conventional methods.

  Preparation of mRNA can be performed by a commonly performed technique. For example, after extracting total RNA from the above-mentioned source by guanidinium thiocyanate-cesium trifluoroacetate method or the like, poly (( A) + RNA (mRNA) can be obtained. Furthermore, poly (A) + RNA may be further fractionated by sucrose density gradient centrifugation or the like.

  After synthesizing single-stranded cDNA using the thus obtained mRNA as a template using oligo dT primer and reverse transcriptase, double-stranded cDNA is synthesized from the single-stranded cDNA. Next, the obtained double-stranded cDNA is incorporated into an appropriate cloning vector to prepare a recombinant vector. A cDNA library can be obtained by transforming Escherichia coli and the like using this recombinant vector, and selecting transformants using tetracycline resistance, ampicillin resistance and the like as indices. In addition, cloning vectors other than plasmids such as λ phage (λgt11 etc.) can also be used.

  In order to select a strain having the target DNA from the transformants obtained as described above, for example, primers designed based on the nucleotide sequence shown in SEQ ID NO: 1 (for example, SEQ ID NO: 3 and 4) are synthesized. A polymerase chain reaction (PCR) can be performed using this, and a method of screening from a cDNA library using the obtained fragment as a probe can be performed.

The amplified DNA fragment thus obtained is labeled with ³² P, ³⁵ S, biotin or the like as a probe, and this is hybridized with a nitrocellulose filter on which the transformant DNA is denatured and immobilized. Screening can be done by searching for strains.

Next, a full-length cDNA is cloned from the obtained clone. For cloning of cDNA, for example, RACE (Rapid Amplification of cDNA ends) method is used. The RACE method is a method for rapidly collecting a 5 ′ or 3 ′ deletion site of cDNA by PCR. The RACE method can also be performed using a commercially available kit (Marathon ^™ cDNA Amplification Kit (Clonetech)).

  The cDNA isolated clone obtained in the above screening is confirmed by determining the cDNA base sequence using the PCR product as a template.

(3) Base sequence and amino acid sequence SEQ ID NO: 1 exemplifies the base sequence of the gene encoding TaMDC1 protein, and SEQ ID NO: 2 exemplifies the amino acid sequence of TaMDC1 protein. The mature form of TaMDC1 protein is presumed to correspond to the 38th to 243rd amino acid sequence of the amino acid sequence shown in SEQ ID NO: 2, and is encoded by the 166th to 786th base sequence of the base sequence shown in SEQ ID NO: 1. In the present invention, as long as the protein containing the amino acid sequence has antibacterial activity, mutations such as deletion, substitution and addition may occur in a plurality of amino acid sequences, preferably one or several amino acids.

  For example, 1 to 10, preferably 1 to 5 amino acids of the 38th to 243rd amino acid sequence of the amino acid sequence shown in SEQ ID NO: 2 may be deleted, and 1 to the amino acid sequence represented by SEQ ID NO: 2 10 to 10 amino acids, preferably 1 to 5 amino acids may be added, or 1 to 10, preferably 1 to 5 amino acids in the amino acid sequence represented by SEQ ID NO: 2 are substituted with other amino acids. May be.

  In order to confirm the presence or absence of antibacterial activity, the TaMDC1 protein may be purified and added to the growth medium of various strains to confirm its growth inhibitory effect. The pathogenic fungi used are not limited, but snow rot fungi such as red snow rot fungus (Monographella nivalis), brown snow rot fungus (Pythium iwayamai, P. paddicum, etc.), snow rot microbe nuclei fungus (Typhula) incarnata, T. ishikarienis) and Sclerotinia borealis.

  Once the base sequence of the gene is determined, the gene is obtained by chemical synthesis, PCR using the cloned cDNA as a template, or hybridization with a DNA fragment having the base sequence as a probe. Can do. Furthermore, a modified nucleic acid encoding TaMDC1 protein can also be synthesized by site-directed mutagenesis or the like. In order to introduce a mutation into a gene, a known method such as Kunkel method or Gapped duplex method or a method based thereon can be employed. For example, mutation is introduced using a commercially available mutation introduction kit using site-directed mutagenesis. Moreover, gene mutation introduction and chimera genes can be constructed by techniques such as error introduction PCR and DNA shuffling. Error introduction PCR and DNA shuffling are techniques known in the art. For example, for error introduction PCR, Chen K, and Arnold FH. 1993, Proc. Natl. Acad. Sci. USA, 90: 5618-5622 See also Stemmer, WP 1994, Nature, 370: 389-391 and Stemmer WP, 1994, Proc. Natl. Acad. Sci. USA 91: 10747-10751 for DNA shuffling.

  Whether or not the recombinant protein produced by the mutated gene has the desired function (antibacterial activity) is expressed in the transformant using the mutated gene, and the antibacterial activity of the resulting recombinant protein is measured as described above. This can be confirmed.

Furthermore, it hybridizes under stringent conditions with a sequence complementary to the whole or a part of the nucleic acid consisting of the 266-786th nucleotide sequence of the nucleotide sequence shown in SEQ ID NO: 1, and has antibacterial activity A gene encoding a protein is also included in the gene of the present invention. Stringent conditions refer to conditions in which specific hybrids are formed and non-specific hybrids are not formed .

(4) Cystatin domain and cystatin-like domain in TaMDC1 The present invention also relates to a polypeptide containing a cystatin domain (domain I) or cystatin-like domain (domain II) of TaMDC1 protein (hereinafter also referred to as cystatin domain polypeptide). Domain I of TaMDC1 protein exhibits cystatin activity, ie, both protease inhibitory activity (papain inhibition) and antibacterial activity. On the other hand, domain II does not show cystatin activity but shows antibacterial activity. Thus, polypeptides comprising TaMDC1 protein domain I and / or polypeptides comprising domain II are also useful. In particular, since a polypeptide containing domain II of TaMDC1 protein exhibits antibacterial activity without inhibiting protease, influences such as indigestion in an organism are avoided when ingested. Therefore, when it is intended to be expressed in a plant, even if the recombinant protein accumulates and remains in the edible part of the plant, it has little effect on the organism and is useful when used as a food or pharmaceutical additive. It is.

  The putative domain I of TaMDC1 protein consists of amino acids 40 to 129 of the amino acid sequence shown in SEQ ID NO: 2, and the putative domain II consists of amino acids 150 to 210 of the amino acid sequence shown in SEQ ID NO: 2. Therefore, the polypeptide of the present invention comprises amino acids 40 to 129 and / or amino acids 150 to 210 of the amino acid sequence shown in SEQ ID NO: 2. However, as long as the polypeptide has antibacterial activity, it may consist of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in these amino acid sequences.

  Nucleic acids encoding TaMDC1 protein domain I and domain II are also provided. Such a nucleic acid is a polypeptide comprising amino acids 40 to 129 of the amino acid sequence shown in SEQ ID NO: 2 or a substitution of one or several amino acids in the amino acid sequence of 40 to 129 of the amino acid sequence shown in SEQ ID NO: 2. , Consisting of a deleted, inserted or added amino acid sequence and encoding a polypeptide having antibacterial activity. For example, nucleic acids encoding TaMDC1 protein domain I include nucleic acids consisting of the 172nd to 441th base sequences of the base sequence shown in SEQ ID NO: 1, and the 172nd to 441st bases of the base sequence shown in SEQ ID NO: 1. A nucleic acid that hybridizes with a nucleic acid comprising a sequence complementary to the sequence under stringent conditions and encodes a polypeptide having antibacterial activity can be exemplified. Moreover, as a nucleic acid encoding TaMDC1 protein domain II, for example, a nucleic acid consisting of the 502-684th base sequence of the base sequence shown in SEQ ID NO: 1, and the 502-684th base sequence of the base sequence shown in SEQ ID NO: 1 Examples thereof include a nucleic acid that hybridizes with a nucleic acid comprising a sequence complementary to the base sequence under a stringent condition and encodes a polypeptide having antibacterial activity.

  Further provided is a reduced domain II that further reduces the scope of domain II and retains antimicrobial activity. In the present specification, “reduced domain II” consists of amino acids 149 to 198 in the amino acid sequence shown in SEQ ID NO: 2. Therefore, the polypeptide of the present invention may contain amino acids 149 to 198 in the amino acid sequence shown in SEQ ID NO: 2. However, as long as the polypeptide has antibacterial activity, it may consist of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in these amino acid sequences.

  Also provided is a nucleic acid encoding the reduced domain II. Such a nucleic acid is a polypeptide comprising amino acids 149 to 198 of the amino acid sequence shown in SEQ ID NO: 2, or one or several amino acids in the amino acid sequence of 149 to 198 of the amino acid sequence shown in SEQ ID NO: 2 , Consisting of a deleted, inserted or added amino acid sequence and encoding a polypeptide having antibacterial activity. For example, the nucleic acid encoding the reduced domain II includes a nucleic acid consisting of the base sequence of 499 to 648 of the base sequence shown in SEQ ID NO: 1 and the base sequence of 499 to 648 of the base sequence shown in SEQ ID NO: 1. And a nucleic acid that hybridizes with a nucleic acid comprising a sequence complementary to the above under stringent conditions and encodes a polypeptide having antibacterial activity.

2. Antibody In the present invention, an antibody against TaMDC1 protein or cystatin domain polypeptide can be prepared and used. In the present invention, the “antibody” means an entire antibody molecule or a fragment thereof (for example, Fab, F (ab ′) ₂ fragment, Fv fragment, etc.) that can bind to a protein that is an antigen or a partial peptide thereof. Or a monoclonal antibody. In the present invention, antibodies (polyclonal antibodies and monoclonal antibodies) can be prepared, for example, by the following method.

  As an immunogen for antibody production, full-length TaMDC1 protein or cystatin domain polypeptide may be used, or a partial peptide thereof may be used. Examples of partial peptides include, but are not limited to, peptides including, for example, amino acid sequences 55 to 70 of the amino acid sequence shown in SEQ ID NO: 2. An immunogen is prepared by dissolving a protein or a partial peptide thereof in a buffer, and if necessary, an adjuvant (complete Freund's adjuvant, incomplete Freund's adjuvant, etc.) may be added for effective immunization. .

  When producing polyclonal antibodies, the immunogen is administered to mammals such as rabbits, rats, mice and the like. Immunization is performed mainly by injecting intravenously, subcutaneously or intraperitoneally. The interval between immunizations is not particularly limited, and immunization is performed 1 to 5 times at intervals of several days to several weeks. Thereafter, after a lapse of a certain period from the last immunization day, the antibody titer is measured by enzyme immunoassay (ELISA, EIA, RIA) or the like, and blood is collected on the day showing the maximum antibody titer to obtain antiserum. Thereafter, the reactivity of the polyclonal antibody in the antiserum is measured by an ELISA method or the like.

  In the case of producing a monoclonal antibody, the immunogen is administered to mammals such as rabbits, rats, mice and the like in the case of polyclonal antibodies. Then, antibody producing cells (spleen cells, lymph node cells, peripheral blood cells, etc.) are collected after a lapse of a certain period from the last immunization day, and a hybridoma is prepared by cell fusion with myeloma cells. In cell fusion, antibody-producing cells and myeloma cells are mixed in an animal cell culture medium that does not contain serum, and a fusion reaction is performed in the presence of a cell fusion promoter (eg, polyethylene glycol). Alternatively, cell fusion can be performed using a commercially available cell fusion device utilizing electroporation.

  The target hybridoma is selected from the cells after cell fusion treatment. In addition, the hybridoma culture supernatant is screened for the presence of antibodies that react with TaMDC1 protein or cystatin domain polypeptide. The screening of hybridomas may be carried out in accordance with a usual method, for example, an enzyme immunoassay method, a radioimmunoassay method or the like can be employed. Cloning of the fused cells is performed by a limiting dilution method or the like to establish a hybridoma that produces the target monoclonal antibody.

  As a method for collecting the monoclonal antibody from the established hybridoma, a normal cell culture method or ascites formation method can be employed. When antibody purification is required in the antibody collection method, known methods such as ammonium sulfate salting-out method, ion exchange chromatography, gel filtration, affinity chromatography are appropriately selected, or a combination thereof is used. Can be purified.

  The antibody against TaMDC1 protein or cystatin domain polypeptide obtained as described above can be used for purification and detection of these proteins.

3. Recombinant vector and transformant The recombinant vector of the present invention can be obtained by ligating (inserting) the gene (nucleic acid) into an appropriate vector. The vector used in the present invention is not particularly limited as long as it can replicate in the host or can integrate the target nucleic acid into the host genome. For example, bacteriophage, plasmid, cosmid, phagemid and the like can be mentioned.

  As plasmid DNA, plasmids derived from actinomycetes (eg, pK4, pRK401, pRF31 etc.), plasmids derived from E. coli (eg, pBR322, pBR325, pUC118, pUC119, pUC18 etc.), plasmids derived from Bacillus subtilis (eg, pUB110, pTP5 etc.) And yeast-derived plasmids (eg, YEp13, YEp24, YCp50, etc.) and the like, and examples of phage DNA include λ phage (λgt10, λgt11, λZAP, etc.). Furthermore, animal viruses such as retrovirus or vaccinia virus, and insect virus vectors such as baculovirus can also be used. In addition, when performing transformation using the Agrobacterium method, a binary vector (pBI121 or the like) can be used.

  In order to insert DNA (nucleic acid sequence) into a vector, first, the purified DNA is cleaved with a suitable restriction enzyme, inserted into a restriction enzyme site or a multicloning site of a suitable vector DNA, and linked to the vector. Is adopted.

  The nucleic acid needs to be incorporated into the vector so that the TaMDC1 protein or cystatin domain polypeptide is expressed. Therefore, in addition to a promoter and a nucleic acid sequence, a cis element such as an enhancer, a splicing signal, a poly A addition signal, a selection marker, a ribosome binding sequence (SD sequence) and the like can be linked to the vector of the present invention as desired.

  A transformant is obtained by introducing a nucleic acid encoding a TaMDC1 protein, a nucleic acid encoding a cystatin domain polypeptide, or a recombinant vector containing these nucleic acids into a host cell so that the protein or polypeptide can be expressed. Obtainable.

  The host used for transformation is not particularly limited as long as it can express a protein or polypeptide. Examples include bacteria (E. coli, Bacillus subtilis, etc.), yeast, plant cells, animal cells (COS cells, CHO cells, etc.) and insect cells.

  When a bacterium is used as a host, the recombinant vector is autonomously replicable in the bacterium, and at the same time, it is composed of a promoter, a ribosome binding sequence, a nucleic acid encoding TaMDC1 protein or cystatin domain polypeptide, and a transcription termination sequence. It is preferable. Examples of Escherichia coli include Escherichia coli DH5α, and examples of Bacillus subtilis include Bacillus subtilis. The method for introducing a recombinant vector into bacteria is not particularly limited as long as it is a method for introducing DNA into bacteria. Examples thereof include a method using calcium ions and an electroporation method.

  When yeast is used as a host, for example, Saccharomyces cerevisiae, Schizosaccharomyces pombe and the like are used. The method for introducing a recombinant vector into yeast is not particularly limited as long as it is a method for introducing DNA into yeast, and examples thereof include an electroporation method, a spheroplast method, and a lithium acetate method.

  When animal cells are used as hosts, monkey cells COS-7, Vero, Chinese hamster ovary cells (CHO cells), mouse L cells, rat GH3, human FL cells and the like are used. Examples of methods for introducing a recombinant vector into animal cells include an electroporation method, a calcium phosphate method, and a lipofection method.

  When insect cells are used as hosts, Sf9 cells and the like are used. Examples of the method for introducing a recombinant vector into insect cells include the calcium phosphate method, lipofection method, electroporation method and the like.

  Further, in the present invention, the plant to be transformed includes the whole plant, plant organs (for example, leaves, petals, stems, roots, seeds, etc.), plant tissues (for example, epidermis, phloem, soft tissue, xylem, fibers). Tube bundle, fence-like tissue, spongy tissue, etc.) or plant cultured cells. The plant used for transformation is not particularly limited, and may be any plant belonging to the monocotyledonous plant class or the dicotyledonous plant class. Examples of plants belonging to the monocotyledonous plant class and the dicotyledonous plant class are shown below, but are not limited thereto.

(Monocotyledonous plant)
Gramineae: Rice (Oryza sativa), Wheat (Triticum aestivum), Barley (Hordeum vulgare), Corn (Zea mays), Perennial ryegrass (Lolium perenne L.), Orchard grass (Dactylis glomerata L.), Meadow fescue (Festuca pratensis Huds) .) Turf grasses such as taros: taro (Colocasia esculenta), konnyaku (Amorphophallus rivieri), pineapples: pineapples (Ananas comosus), etc .: coconuts (Cocos nucifera), lilies: leeks (Allium fistulosum), onions (Allium cepa), Garlic (Allium sativum), Asparagus (Asparagus officinalis), Katakuri (Erythronium Japonicum), etc.

(Dicotyledonous plant)
Rabbitaceae: Spinach (Spinacia oleracea), sugar beet (Beta vulgaris), etc. ), Rapeseed (Brassica campestris L., B. napus L.) and other cucurbitaceae: cucumber (Cucumis sativus), melon (Cucumis melo), watermelon (Citrullus lanatus) and other asteraceae: lettuce (Lactuca sativa), burdock (Arctium lappa) ), Safflower (Carthamus tinctorius), etc. Pepperaceae: Pepper nigrum, Lamiaceae: Sage (Salvia officinalis), Basil (Ocimum basilicum), Solanum: Solanum melongena, Potato (Solanum tuberosum), Tomato (Lycopersicum) esculentum), peppers (Capsicum annuum), tobacco (Nicotiana tabacum), etc. Rosaceae: Prunus mume, peach (Prunus persica), apple (Maluspumi) la var.domestica), Strawberries (Fragaria x ananassa), etc. Convolvulaceae: Sweet potatoes (Ipomonea batatas), etc. Grapes: Grapes (Vitis vinifera, Vitis labrusca), etc. Legumes: Peas sativum, Broad bean (Vicia faba), Groundnut (Arachis hypogaea), soybean (Glycine max), azuki (Vigna angularis), etc. Citrus unshiu, grapefruit (Citrus paradisi), lemon (Citrus limon) and the like.

  The plant to be transformed in the present invention is preferably a plant belonging to the family Gramineae, particularly a plant that is desired to impart resistance to snow rot fungi, such as wheat, barley, and turfgrass.

  The recombinant vector can be introduced into plants by a conventional transformation method such as the Agrobacterium method, particle gun method, PEG method, electroporation method and the like. For example, when the Agrobacterium method is used, the constructed plant expression vector is introduced into an appropriate Agrobacterium such as Agrobacterium tumefaciens, and this strain is introduced by the leaf disc method (Hirofumi Uchimiya, plant gene). According to the operation manual, 1990, 27-31pp, Kodansha Scientific, Tokyo, etc., a sterile cultured leaf piece can be infected to obtain a transformed plant.

  When the particle gun method is used, a plant body, a plant organ, and a plant tissue itself may be used as they are, or may be used after preparing a section, or a protoplast may be prepared and used. The sample thus prepared can be processed using a gene introduction apparatus (for example, PDS-1000 (BIO-RAD)). The treatment conditions vary depending on the plant or sample, but are usually performed at a pressure of about 450 to 2000 psi and a distance of about 4 to 12 cm.

  When plant cultured cells are used as a host, transformation is performed by introducing a recombinant vector into the cultured cells by the particle gun method, electroporation method or the like.

  Callus, shoots, hairy roots, etc. obtained as a result of transformation can be used as they are for cell culture, tissue culture or organ culture, and can be used at a suitable concentration using conventionally known plant tissue culture methods. The plant can be regenerated by administration of plant hormones (auxin, cytokinin, gibberellin, abscisic acid, ethylene, brassinolide, etc.).

  Whether or not a gene has been introduced into the host can be confirmed by PCR, Southern hybridization, Northern hybridization, or the like. For example, DNA is prepared from the transformant, PCR is performed by designing a DNA-specific primer. PCR can be performed under the same conditions as those used for preparing the plasmid. Thereafter, the amplification product is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis, etc., stained with ethidium bromide, SYBR Green solution, etc., and the amplification product is detected as one band, It can be confirmed that it has been transformed. Moreover, PCR can be performed using a primer previously labeled with a fluorescent dye or the like to detect an amplification product. Furthermore, it is possible to employ a method in which the amplification product is bound to a solid phase such as a microplate and the amplification product is confirmed by fluorescence or enzyme reaction. Or you may perform the selection of the transformant by a chemical | medical agent.

4). Production of Protein / Polypeptide The TaMDC1 protein and cystatin domain polypeptide of the present invention can be produced from the aforementioned wheat by protein purification methods known in the art. It can also be produced by chemical peptide synthesis methods known in the art, such as solid phase synthesis. It can also be produced by culturing a transformant introduced with a nucleic acid encoding TaMDC1 protein or cystatin domain polypeptide and recovering the protein from the resulting culture. Protein production methods using such genetic engineering techniques are well known in the art and will be described in detail below.

  The TaMDC1 protein and cystatin domain polypeptide of the present invention can be obtained by culturing the transformant and collecting it from the culture. “Culture” means any of culture supernatant, cultured cells, cultured cells, or disrupted cells or cells. The method for culturing the transformant of the present invention is carried out according to a usual method used for culturing a host.

  A medium for culturing transformants obtained using microorganisms such as Escherichia coli and yeast as a host contains a carbon source, nitrogen source, inorganic salts, etc. that can be assimilated by the microorganisms, and efficiently cultures the transformants. As long as the medium can be used, either a natural medium or a synthetic medium may be used. Examples of the carbon source include carbohydrates such as glucose, fructose, sucrose, and starch, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol. Examples of the nitrogen source include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium salts of organic acids such as ammonium phosphate or other nitrogen-containing compounds, peptone, meat extract, corn steep liquor, and the like. Examples of the inorganic substance include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.

  The culture is usually performed at 20 to 37 ° C. for 12 hours to 3 days under aerobic conditions such as shaking culture or aeration and agitation culture.

Examples of a medium for culturing a transformant obtained using animal cells as a host include generally used RPMI 1640 medium, DMEM medium, or a medium obtained by adding fetal calf serum or the like to these mediums. The culture is usually performed at 37 ° C. for 6 hours to 4 days in the presence of 5% CO ₂ .

  After culturing, when the protein or polypeptide of the present invention is produced in cells or cells, the protein or polypeptide is subjected to ultrasonic treatment, repeated freeze-thawing, homogenizer treatment, etc. to disrupt the cells or cells. Collect. When the protein is produced outside the cells or cells, the culture solution is used as it is, or the cells or cells are removed by centrifugation or the like. Thereafter, by using general biochemical methods used for protein isolation and purification, such as ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc. alone or in appropriate combination, From the above, the protein of the present invention can be isolated and purified.

5). Resistant plants When a gene encoding the TaMDC1 protein, a gene encoding the cystatin domain polypeptide of TaMDC1 or a recombinant vector is introduced into the plant, the resulting transformed plant has resistance to pathogenic bacteria, particularly snow rot fungi. To win. Therefore, the transformed plant can be used as a pathogen-resistant plant. Here, “pathogenic bacteria” means bacteria, fungi, and the like that cause plant diseases. In the present invention, the pathogenic bacteria are not particularly limited as long as they are bacteria and fungi. For example, fungi such as snow rot fungus, rice rot fungus (Magnaporthe grisea), wheat red mold fungus (Fusarium graminearum), and rice that is a bacterium. An example is Xanthomonas oryzae. Snow rot fungi include, for example, red snow rot fungus (Monographella nivalis), brown snow rot fungus (Pythium iwayamai, P. paddicum, etc.), snow rot fungus (Typhula incarnata, T. ishikarienis), snow rot fungus It contains disease bacteria (Sclerotinia borealis). In addition, the resulting transformed plants can also acquire resistance to abiotic stresses such as low temperature, salt and drought. Here, “resistance” means resistance to pathogenic bacteria and abiotic stress, that is, by reducing the influence of pathogenic bacteria or abiotic stress or not being affected at all, the plant is alive and nourishing. It means that growth and reproductive growth can be continued.

  The pathogen resistance of the transformed plant of the present invention can be assayed by the following method. For example, the leaves of the resulting transformed plant are scratched with a needle or the like (Mock treatment), inoculated with a bacterial solution containing a pathogenic bacterium, and the area of the part damaged by the infection is treated similarly. The resistance of the transformed plant can be assayed by comparing with the above. It will be apparent to those skilled in the art that the assay conditions may vary depending on the plant species and pathogen species being assayed. More easily by staining with dyes such as aniline blue (Eschrich W. and Currier HB, (1964) Stain Technology 39, 303-307) after fixation of tissue or cells or in a live state during the assay. Can be compared. Alternatively, seeds obtained from a transformed plant are immersed in a pathogenic fungus solution, sown in soil, germinated and grown, and the number of individuals that grow normally and the number of individuals that grow from the seeds of a non-transformed plant treated in the same manner It can also be examined by comparing In addition, the transformed plant cells and non-transformed plant cells may be cultured in a medium to which a pathogenic bacterium is added for a predetermined period (for example, 6 hours to 5 days), and assayed by comparing the number of surviving cells. The method for assaying disease resistance of the transformed plant of the present invention is not limited to the above.

  In addition, the abiotic stress resistance of the transformed plant of the present invention was treated in the same manner as the normal number of individuals by germination, growth or flower bud formation in the presence of abiotic stress. It can be examined by comparing the number of non-transformed plants.

  The resistant plant of the present invention is a transgenic plant into which a nucleic acid encoding TaMDC1 protein or a nucleic acid encoding a cystatin domain polypeptide or a recombinant vector containing these nucleic acids is introduced as a resistant plant. It can be produced by breeding to such an extent that it can be used. In this case, it is only necessary to select a plant that exhibits resistance to the plant without causing death or the like under the conditions causing the various diseases or abiotic stress. The start of use as a resistant plant may be immediately after transformation or after culturing or cultivating for 1 to about 90 days after transformation.

  Moreover, the resistant plant of the present invention forms seeds for propagation by crossing (for example, self-pollination, pollination between transformed plants, or pollination between transformed plants and non-transformed plants). Can do.

  Furthermore, using the above-described procedure for differentiation / induction of plant bodies from infected cells, tissue culture of transformed plant tissues (eg, roots, stems, leaves) or organs (eg, growth points, pollen) By means of this, further transformed plants can be obtained without going through the reproductive process (seed). Such techniques and procedures are known to those skilled in the art, and general methods of tissue culture are described in various experimental manuals.

  The resistant plant of the present invention thus obtained grows normally and exhibits high resistance to pathogenic bacteria or abiotic stress by expression of TaMDC1 protein or cystatin domain polypeptide. In addition, seeds obtained from transformed plants also germinate and grow normally and show high resistance. This indicates that the introduced nucleic acid or recombinant vector is preserved in the next generation, and thus the resistance is stably inherited to progeny. Therefore, according to the present invention, a practical and useful plant exhibiting high resistance can be obtained.

6). Antibacterial agent TaMDC1 protein and the CY domain polypeptide of the protein have antibacterial activity against pathogenic bacteria. Therefore, TaMDC1 protein and cystatin domain polypeptide, nucleic acid encoding the protein or polypeptide, recombinant vector containing the nucleic acid, transformant, and the like can be used as an antibacterial agent.

  The antibacterial agent which concerns on this invention may contain only 1 type among said protein or polypeptide, a nucleic acid, a recombinant vector, or a transformant, and may contain multiple types. The antibacterial agent of the present invention may further contain other antibacterial agents and / or additives. Any other antibacterial agents and additives may be used as long as they do not reduce the antibacterial activity of the components contained in the antibacterial agent of the present invention.

  The form of the antibacterial agent of the present invention is not particularly limited, and it can be prepared as a solution by dissolving or suspending TaMDC1 protein or cystatin domain polypeptide in a suitable solvent, or as a powder or tablet. A culture obtained by culturing the above transformant can also be used as it is as the antibacterial agent of the present invention.

  Although the solvent which can be used in a liquid agent is also not specifically limited, For example, water, methanol, dimethylsulfoxide etc. are mentioned. When preparing as a liquid agent, the concentration of the active ingredient is not particularly limited as long as it is in a range where antibacterial activity can be exhibited. When the antibacterial agent of the present invention is prepared as a powder or tablet, in addition to the protein, polypeptide, nucleic acid, etc., powders known to those skilled in the art such as inorganic salts, saccharides, talc, vermulite, etc. And mixed to form a powder or tablet.

  The target to which the antibacterial agent of the present invention is applied is not particularly limited, but is a plant in which pathogenic bacteria are generated (for example, a plant belonging to the family Gramineae, preferably wheat, barley, turf, etc.), a field where the plant grows. , Gardens and lakes.

  The antibacterial agent of the present invention can be used by spraying directly on a target plant, or spraying directly on an area, or immersing a container growing a plant in the antibacterial agent. The amount used depends on the type of target and the surrounding environment.

  Bacteria and fungi are included as pathogenic bacteria that are sterilized by the antibacterial agent of the present invention or whose growth is suppressed. The antibacterial agent of the present invention is particularly effective against snow rot fungi that are filamentous fungi. Snow rot fungi include, for example, red snow rot fungus (Monographella nivalis), brown snow rot fungus (Pythium iwayamai, P. paddicum, etc.), snow rot fungus (Typhula incarnata, T. ishikarienis), snow rot fungus It contains disease bacteria (Sclerotinia borealis).

  The antibacterial agent of the present invention is a protein derived from a protein derived from wheat and a domain of the protein, and is expected to be widely used in the agricultural and pharmaceutical fields as an antibacterial agent that is environmentally friendly and excellent in safety.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, the technical scope of this invention is not limited to these Examples.

In this example, an attempt was made to isolate an antibacterial protein from wheat.
(1) Plant material and low temperature acclimation The surface of winter wheat (Triticum aestivum L. cv. Chihoku) was sterilized and planted in a commercial soil mixture after germination. The plants were grown for 14 days in a growth room at 22 ° C./18° C. (16 hours illumination / 8 hours dark room). Cold acclimation was carried out at 6 ° C./2° C. (8 hours illumination / 16 hours dark room) for another 2 weeks. In order to cancel the low-temperature acclimation, the film was grown at 22 ° C./18° C. (16 hours illumination / 8 hours dark room) for 3 days.

  Crown and leaf tissues were pre-acclimated (non-acclimated (NA), day 0), cold acclimatized (CA) 1, 3, 7, 10, 14 days, deacclimated (DA) 1 And on day 3. The collected plant material was frozen in liquid nitrogen and stored at −80 ° C. until RNA and protein extraction.

(2) Isolation and sequence analysis of cDNA Screening of cold-regulated cDNA clones in a cDNA library constructed from cold-adapted coronary tissue was performed using the macroarray technique.

  More specifically, total RNA was extracted from the crown tissue of winter wheat (Triticum aestivum L. cv. Chihoku) that had been acclimated by the above-described method using TRIzol reagent (Invitrogen), and Dynabeads (DYNAL) was used. mRNA was purified. A cDNA library was prepared by the manufacturer-specified method using ZAP express cDNA synthesis Kit (Stratagene).

Plasmid DNA from cDNA clones was denatured and slot blotted onto Hybond N ⁺ membranes (Amersham Biosciences) using a vacuum immunoblotter (Atto, Tokyo, Japan). Subsequently, the membrane was UV fixed, hybridized with double stranded cDNA synthesized from mRNA isolated from NA and CA crown tissue, and labeled with Alk Phos Direct Kit (Amersham Biosciences). Hybridization and membrane washing were performed according to the manufacturer's protocol. Signal detection and quantification were performed with an LAS3000 image analyzer (Fuji Film, Tokyo, Japan) using Array Grandeur software. Clones up-regulated by low temperature were sequenced with DNA sequencer 373A (Applied Biosystem, San Jose, Calif.) Using Big Dye Terminator Cycle Sequencing Kit v 1.1 v 3.1 (Applied Biosystem). Analysis of sequencing data was performed with STADEN PAKAGE software and sequence alignment was performed with CLUSTAL X. Multigene phylogenetic trees were determined using the neighborhood joining method and displayed with the Tree View software.

  The gene of the obtained clone was found to belong to a new class of plant cystatin family by BLASTX search. This gene was named TaMDC1. The base sequence and deduced amino acid sequence of the cDNA clone are shown in FIG. 1 and SEQ ID NOs: 1 and 2, respectively. In FIG. 1, the underlined codon indicates a translation start codon, and the asterisk indicates a stop codon. The full-length cDNA clone contains 998 nucleotides, does not contain a poly A tail, has an open reading frame of 731 nucleotides, and has a putative polyadenylate addition signal in the 3 ′ untranslated region (underlined in the base sequence of FIG. 1). portion). The encoded 243 amino acid protein has a predicted N-terminal secretion signal of 37 amino acids (underlined portion in the amino acid sequence of FIG. 1). The estimated molecular weight of the precursor protein is 26,477 Da, and the mature protein is 23,325 Da.

When a CDD search (Marchler-Bauer, A., JB Anderson, et al. (2005). Nucleic Acids Res 33 Database Issue: D192-6) was performed using the deduced amino acid sequence, a conserved cystatin-like domain ( Two domains showing homology with the CY domain (smart00043) were identified (shown in gray background in FIG. 1). The first putative domain (# 40-129, FIG. 1) showed 99.1% identity when aligned with the CY domain. Further, ^{Gly 43,} the reaction site motif ^{QXVXG (Q 86 T 87 V 88} A 89 G 90), W 117 motifs, and advanced as required to cysteine protease inhibitory activity, including LARFAV motifs like that are highly conserved in phyto cystatin All of the conserved motifs were included (Arai, S., et al. 1991, J. Biochem. (Tokyo) 109: 294-8; Margis et al. 1998, Arch Biochem Biophys 359: 24-30). The second putative domain of TaMDC1 (Nos. 150-210, FIG. 1), which forms the C-terminal extension, only partially matches the conserved CY domain (57%) and is conserved as described above. No motif was found in this domain.

  A BLASTP search was performed using the deduced amino acid sequence of the TaMDC1 protein (SEQ ID NO: 2), revealing homology with other plant cystatins. TaMDC1 has some degree of homology with the unnamed rice-derived cystatin-like protein (GenBank accession number NP — 912935) and shows 89% identity. The result of comparing TaMDC1 with a known plant cystatin is shown in FIG. In FIG. 2, black marks indicate identical residues, and gray marks indicate partially conserved residues. The alignment shown in FIG. 2 was performed with the ClustalW program and colored using GeneDoc software.

  As shown in FIG. 2, soybean cystatin (BAA19608) and 67%, apple cystatin (AAO19652) and 68%, taro-derived cystatin (AAM88397) and 60%, and cabbage cystatin (AAC37479) and 64% identity (64%), respectively. Figure 2). The homology between rice-derived cystatin and TaMDC1 is 59% identity (71% similarity) with oryzastatin I (OCI; AAA33903) and 53% identity (70% with oryzasistatin II (OCII; AAA33911) (Fig. 2). The identity of TaMDC1 with other reported wheat cystatins is 54%, 55%, 55% and 47% with wheat cystatin WC1 (BAB18766), WC2 (BAB18769), WC3 (BAB18768) and WC4 (BAB18767). And has only 32% identity with WC5 (AAL56401). TaMDC1 has 70%, 73%, 71%, 62% and 51% homology with WC1 to WC5, respectively.

  In this example, the gene encoding TaMDC1 protein was used for recombinant expression in E. coli.

  PCR amplification of TaMDC1 cDNA clones was performed using specific primers incorporating BamHI and XmaI sites for cloning into the pGEX6P-3 expression vector (Amersham Biosciences, UK). The primers used were a sense primer (5′-GGATCC GCCATGGCCAGCCACGTCCT-3 ′; SEQ ID NO: 3) and an antisense primer (5′-CCCGGG TCACTGGCTGCTAGATTCGT-3 ′; SEQ ID NO: 4). The resulting construct was used for transformation of E. coli BL21 (DE3) strain. Purification of the recombinant protein (GST-TaMDC1) was performed on a glutathione-Sepharose 4B column (Amersham Biosciences, UK), and the fusion GST-TaMDC1 was purified at 4 ° C. using PreScission® protease (Amersham Biosciences). Digested for 16 hours (GST was excised). The purified protein was concentrated with a Microcon YM-10 column (Millipore Corporation, USA) and the concentration was measured using a BioRad Protein Assay Kit (BioRad Laboratories, USA) with BSA as a reference. Protein samples were separated by SDS-PAGE and stained with Coomassie blue dye using standard protocols.

  The result is shown in FIG. In FIG. 3, lane 1 shows the total protein fraction, lane 2 shows the total protein fraction 1 hour after IPTG induction, lane 3 shows the soluble fraction, lane 4 shows purified cystatin, and lane M shows the low molecular weight peptide standard.

A putative mature form of TaMDC1 (mTaMDC1) lacks the putative hydrophobic signal sequence (Met ¹ -Ala ³⁵ in SEQ ID NO: 2) and was expressed in E. coli BL21 (DE3) as an N-terminal GST fusion in the pGEX6P3 vector. For expression of mTaMDC1, an expression vector was designed based on the amino acid sequence of 36 to 243 of SEQ ID NO: 2 including a putative mature portion (amino acids 38 to 243 of SEQ ID NO: 2). Expression of the GST-TaMDC1 fusion protein was induced by the addition of IPTG (FIG. 3, lane 2). Most of the GST-mTaMDC1 fusion protein was detected in the soluble fraction (FIG. 3, lane 3). SDS-PAGE analysis also showed the presence of a single band of purified TaMDC1 protein with a molecular weight of about 28 kDa (FIG. 3, lane 4).

  In this example, an enzyme inhibition assay of a mature TaMDC1 protein (mTaMDC1) and a Ki value were measured.

  Enzyme inhibition of papain (Sigma Chemical Co.) by mTaMDC1 (containing amino acids 38-243 in SEQ ID NO: 2) is shown by Yosa (Yoza, K. et al. (2002). Biosci Biotechnol Biochem 66: 2287-91). Was performed using BAPA (benzoyl-L-arginine-p-nitroanilide; Peptide Institute Inc., Osaka, Japan) as a substrate. Briefly, the inhibitory activity of various concentrations of mTaMDC1 on papain (0.1 mg / ml) was determined using BAPA (100 mM sodium phosphate, pH 6.8, 2.5 mM DTT, 10 mM EDTA) using BAPA ( 10 mM) as a substrate. Enzyme activation and incubation with BAPA were performed at 37 ° C. for 15 minutes in each step. The absorbance of the enzyme reaction was measured at 405 nm using a BioRad microplate reader. The enzyme inhibition reaction was tested three times for each concentration of mTaMDC1 analyzed. The Ki value of mTaMDC1 for papain was calculated from the LB plot using Excel software.

  The results are shown in FIG. FIG. 4 represents the inhibitory activity of mTaMDC1 on papain as the residual enzyme activity at various concentrations of inhibitor when BAPA is used as a substrate. In FIG. 4, the vertical line represents the standard deviation. When enzyme titration was performed with increasing concentrations of mTaMDC1, papain lost almost all activity in equimolar amounts of cystatin, suggesting that the mechanism of interaction is 1: 1. (FIG. 4).

The mTaMDC1 Ki value for papain was calculated from inhibition curves of 4 different substrate concentrations and 3 inhibitor concentrations. The measured value of Ki was 5.8 × 10 ⁻⁷ M for mTaMDC1, comparable to other plant cystatins (10 ⁻⁷ to 10 ⁻⁹ M).

In this example, the expression of TaMDC1 protein in plants was observed.
(1) Northern blot analysis Total RNA was isolated from the wheat prepared in Example 1 and subjected to Northern blot analysis. Total RNA was isolated from wheat shoots, roots and crown tissue using TRIzol reagent (Invitrogen). Total RNA (20 μg) was separated on a 1.2% agarose gel containing formaldehyde and transferred onto Hybond N + membranes (Amersham Biosciences). Hybridization was performed using full-length TaMDC1 cDNA (base sequence 1 to 1031 in SEQ ID NO: 1) labeled with an ECL kit (Amersham Biosciences) according to the manufacturer's instructions. Signal detection and quantification were performed with a LAS3000 image analyzer (Fuji Film, Tokyo, Japan).

  The results are shown in FIG. In FIG. 5, a shoot tissue (0 to 48 hours) subjected to a low temperature treatment (4 ° C.) for a short period, a low temperature acclimation (0 to 14 days) and a deacceleration (22 ° C., +1, +3). Shows the coronary tissue on which TaMDC1 accumulation showed maximum induction at 72 hours and was strongly induced by low temperature treatment in shoots and coronary tissue (FIG. 5). In addition, the expression level returned to the level of the untreated control 1 day after deacclimation (FIG. 5, right side).

(2) Western blot analysis The NA, CA and DA wheat crown and leaf tissues prepared in Example 1 were ground with liquid nitrogen, and total protein was added to sodium phosphate buffer (PMSF (1 mM) and PVPP added) ( Extraction was performed using 50 mM, pH 7.5). After centrifugation, the protein concentration in the supernatant was measured using the BioRad protein assay kit (BioRad Laboratories, USA) using BSA as a reference. The extracted total protein (50 μg) was separated by SDS-PAGE and transferred onto Hybond-C extra membrane (Amersham Biosciences). Hybridization was performed using polyclonal rabbit serum raised against recombinant mTaMDC1 (1: 5000 v / v) and anti-rabbit IgG peroxidase-conjugated antibody (1: 100,000 v / v) (Amersham Biosciences). The anti-TaMDC1 antibody was used to immunize rabbits using recombinant mTaMDC1 purified from E. coli as an antigen to obtain antiserum.

  Signal detection was performed using the ECL system (Amersham Biosciences) according to the manufacturer's instructions.

  The results are shown in FIG. FIG. 6 shows the results of the crown and leaf tissues of wheat acclimated at low temperatures (4 ° C., 0-14 days) and deacclimated (22 ° C., +1, +3 days). Cold induced accumulation of TaMDC1 protein was shown by Western blot analysis with polyclonal antisera raised against purified mTaMDC1 (FIG. 6). A clear induction was detected on day 1 and maximal protein accumulation was observed on days 7 and 10 of cold acclimation in the coronary tissue (FIG. 6, bottom). Protein expression returned to control levels on day 3 of deacclimation at 22 ° C. TaMDC1 protein was less accumulated in leaf tissue compared to the crown (FIG. 6, bottom). Nevertheless, clear induction was also observed in the leaves after 3 days of treatment.

In this example, the expression of TaMDC1 protein by abiotic stress treatment in plants was observed.
(1) Abiotic stress treatment Germinated seeds of winter wheat (Triticum aestivum L. cv. Chihoku) were hydroponically grown on a plastic mesh lattice filled in a container with distilled water. Seedlings were grown in a growth room for 7 days at 22 ° C. under continuous lighting. Subsequently, the plants are transferred to plastic containers containing cold water (4 ° C.), 50 μM abscisic acid (ABA), or 200 mM NaCl, or no water, respectively, and various abiotic stresses (low temperature, ABA, salt, dry) The plants were tested for. Shoot and root tissues were collected before treatment (0 hours) and 1, 2, 4, 6, 10, 24 and 48 hours after treatment with the respective stress. Samples were frozen in liquid nitrogen and stored at −80 ° C. until RNA and protein extraction.

(2) Northern blot analysis The expression pattern of TaMDC1 in various abiotic stress treatments was examined by Northern blot analysis. Total RNA was isolated from wheat shoots and roots using TRIzol reagent (Invitrogen). Total RNA (20 μg) was separated on a 1.2% agarose gel containing formaldehyde and transferred onto Hybond N + membranes (Amersham Biosciences). Hybridization was performed using full length TaMDC1 cDNA labeled with an ECL kit (Amersham Biosciences) according to the manufacturer's instructions. Signal detection and quantification were performed with a LAS3000 image analyzer (Fuji Film, Tokyo, Japan).

  The results are shown in FIG. In FIG. 7, the gel stained with ethidium bromide is the same RNA sample introduced. Accumulation of TaMDC1 mRNA was induced in the roots at 6 hours by ABA treatment, but the expression level in shoots remained unchanged (FIG. 7, upper panel). In contrast to ABA, salt and drying treatments induced TaMDC1 message expression in both shoot and root tissues (FIG. 7, middle and bottom). However, the response at the root was earlier and more pronounced. Significant induction of TaMDC1 message was observed in the roots as in 2 hours of salt treatment, whereas in shoots, induction was detected 12 hours after treatment (FIG. 7, middle panel). Similarly, by dry treatment, an increase in expression was detected 10 hours after treatment in the roots and 24 hours in shoot tissues (FIG. 7, lower panel). Therefore, TaMDC1 protein is thought to be involved in acquiring resistance to abiotic stresses such as low temperature, salt, abscisic acid and drought in plants.

In this example, an antibacterial activity assay of mTaMDC1 was performed.
A phytopathogenic fungus (Microdochium nivale MCW222-7 strain) was cultured on PDA (Potato Dextrose Agar, Difco) at 15 ° C. for 1-2 weeks. Fungal hyphae were extracted with water and used in antimicrobial activity assays by two parallel experiments after cleavage with small colony forming units (cfu). .

The inhibitory activity of mTaMDC1 is shown in PDB (Potato Dextrose Broth, Difco), M.M. Tested in tubes (total volume 1 ml) containing nivale mycelium (10 ³ to 10 ⁴ cfu / ml) and various amounts of mTaMDC1 (0, 10, 20, 30, 40 and 50 μg / ml). The test tube was incubated at 15 ° C. for 7 days, and mycelial growth was observed directly and with a microscope (OLYMPUS BX51). Samples for microscopic observation of mycelium were stained with Lacto-Fuchine and 400 × magnification was used.

Relative fungal growth was performed on a microplate containing PDB (total volume 200 μl) and various amounts of mTaMDC1 (0, 10, 20, 30, 40 and 50 μg / ml). The growth was assessed by the growth of nival mycelium (10 ³ to 10 ⁴ cfu / ml). Mycelial growth was monitored by measuring absorbance at 492 nm (BioRad Microplate Reader) after incubation at 15 ° C. for 7 days to evaluate relative mycelial growth. The experiment was performed three times.

  The results are shown in FIGS. FIG. 8 shows a photograph of the mycelium grown in each test tube. In the absence of mTaMDC1 and in the presence of mTaMDC1 up to 30 μg / ml, vigorous hyphal growth was observed. Mycelial growth in test tubes was markedly inhibited when 40 μg / ml mTaMDC1 was used, and no hyphal growth was observed in the samples using 50 μg / ml mTaMDC1 (FIG. 8).

  FIG. 9 shows the antibacterial activity of various concentrations of mTaMDC1 as a percentage (%) of mycelial growth in the absence of mTaMDC1. The vertical axis represents standard deviation. The inhibitory activity of mTaMDC1 protein was 100% with 50 μg / ml mTaMDC1 (FIG. 9). Mycelium growth in control tubes in the absence of cystatin and in cultures with 10, 20, 30, and 40 μg / ml mTaMDC1 was 102% -15%, 100%, 102%, 93%, respectively. 68% and 15% (FIG. 9).

  FIG. 10 shows the results of observing the antibacterial activity of mTaMDC1 at various concentrations with a microscope. In FIG. 10, the M.M. in control tube (1), in the presence of 10 μg / ml mTaMDC1 (2), and in the presence of 50 μg / ml mTaMDC1 (3). nivale mycelium is shown. In a control medium without mTaMDC1 (1 in FIG. 10), mycelia grow and It became a mycelium of a form typical for the nivale. Similar fungal growth was observed in media supplemented with 10, 20, and 30 μg / ml mTaMDC1 (FIG. 10-2). In the presence of high concentrations of mTaMDC1, mycelial development was markedly inhibited. Hyphae morphological changes (such as bumps and fragmentation) were observed in samples containing 40 and 50 μg / ml mTaMDC1 (3 in FIG. 10). Importantly, inhibitory concentrations of mTaMDC1 also induced sporulation in addition to the above morphological changes.

In this example, a polypeptide containing TaMDC1 protein domains I and II was prepared and its antibacterial activity was confirmed.
(1) Preparation of polypeptide (Met36-Thr133) containing domain I (40-129) Primer d1BmF (5'-GCGCGCGGATCCATGGCCAGCCACGTCCT-3 '; SEQ ID NO: 5) and d1XmR (5'-GCGCGCCCCGGGTCAGGTGGCGTCCCCGGT) using TaMDC1 full-length cDNA as a template The fragment amplified by PCR using -3 ′; SEQ ID NO: 6) was digested with BamHI and XmaI and then cloned into the expression vector pGEX6P-3 (Amersham Biosciences, UK). Thereafter, as described in Example 2, induction and purification were performed in the same manner as mTaMDC1.

(2) Preparation of polypeptide (Asp131-Q243) containing domain II (150-210) Primer d2BmF451 (5'-GCGCGCGGATCCGACGCCACCTCCTTCACC-3 '; SEQ ID NO: 7) and d2XmR798 (5'-GCGCGCCCCGGGTCAGATTCACTGGCTGCT) using TaMDC1 full-length cDNA as a template The fragment amplified by PCR using -3 ′; SEQ ID NO: 8) was digested with BamHI and XmaI and then cloned into the expression vector pGEX6P-3. Thereafter, as described in Example 2, induction and purification were performed in the same manner as mTaMDC1.

(3) Confirmation of papain inhibitory activity In the same manner as described in Example 3, the papain inhibitory activity of the polypeptide comprising domain I or II prepared in (1) and (2) was confirmed. The results are shown in FIG. As a result, it was found that the polypeptide containing domain I exhibits papain inhibitory activity in a concentration-dependent manner, whereas the polypeptide containing domain II does not show papain inhibitory activity even in the presence of a high concentration.

(4) Confirmation of antibacterial activity The antibacterial activity of the polypeptide containing domain I or II prepared in (1) and (2) was confirmed by the same method as described in Example 6. The results are shown in FIG. As a result, both polypeptides comprising domain I and polypeptides comprising domain II are It was shown to have hyphal growth inhibitory activity (antibacterial activity) in a concentration-dependent manner with respect to the nival.

(5) Preparation of polypeptide containing reduced domain II (Pro149-Phe198) Using TaMDC1 full-length cDNA as a template, primers d2_67BmF (5'-GCGCGCGGATCCATGCCTGGATGGCGTGA-3 '; SEQ ID NO: 9) and d2_217XmR (5'-GCCGCGCCCCGGGTTAGAAGTCTTCAACC-3' A fragment amplified by PCR using SEQ ID NO: 10) was digested with BamHI and XmaI and then cloned into the expression vector pGEX6P-3. Thereafter, as described in Example 2, induction and purification were performed in the same manner as mTaMDC1, and a polypeptide (mDII) containing the reduced domain II was obtained.

(6) Confirmation of antibacterial activity The antibacterial activity of the polypeptide (mDII) containing the reduced domain II prepared in (5) was confirmed by the same method as that described in Example 6. The results are shown in FIG. As a result, a polypeptide comprising reduced domain II is It was shown to have hyphal growth inhibitory activity (antibacterial activity) in a concentration-dependent manner with respect to the nival.

  The present invention provides a protein (polypeptide) that imparts antibacterial activity to various organisms including plants and a gene encoding the same. Moreover, by using the protein and a gene encoding the protein, a plant (Gramineae plant, particularly wheat) having improved resistance to pathogenic bacteria and abiotic stress can be produced. In addition, an antibacterial agent using the protein and a gene encoding the protein is also provided.

The base sequence (SEQ ID NO: 1) and deduced amino acid sequence (SEQ ID NO: 2) of the nucleic acid encoding the wheat cystatin protein TaMDC1 are shown. The result of having performed arrangement | sequence arrangement | sequence with wheat cystatin protein TaMDC1 and various plant cystatin proteins is shown. It is the result of SDS-PAGE which shows the expression in Escherichia coli of recombinant mTaMDC1 protein. It is a graph which shows the papain inhibitory activity of mTaMDC1. It is the result of the Northern blot analysis about TaMDC1 gene expression. It is the result of the Western blot analysis about TaMDC1 expression. FIG. 6 is a result of Northern blot analysis of TaMDC1 gene expressed in response to ABA, salt and desiccation. A photograph of mycelium grown in a test tube is shown. The percentage of antibacterial activity of mTaMDC1 at various concentrations to mycelial growth in the absence of mTaMDC1 is shown. The result of having observed the antimicrobial activity of mTaMDC1 in various density | concentrations with the microscope is shown. It is a graph which shows the papain inhibitory activity of the polypeptide containing domain I and the polypeptide containing domain II. 2 shows the antimicrobial activity of a polypeptide comprising domain I and a polypeptide comprising domain II. FIG. 5 shows the antimicrobial activity of a polypeptide (mDII) containing a reduced domain II.

  Sequence numbers 3-10: Synthetic oligonucleotide

Claims (25)

The following polypeptide (a) or (b).
(A) Polypeptide consisting of amino acids 149 to 198 of the amino acid sequence shown in SEQ ID NO: 2 (b) One or several amino acids are substituted in the amino acid sequence of 149 to 198 of the amino acid sequence shown in SEQ ID NO: 2 , A polypeptide comprising an amino acid sequence deleted, inserted or added and having antibacterial activity without inhibiting protease A nucleic acid encoding the following polypeptide (a) or (b).
(A) Polypeptide consisting of amino acids 149 to 198 of the amino acid sequence shown in SEQ ID NO: 2 (b) One or several amino acids are substituted in the amino acid sequence of 149 to 198 of the amino acid sequence shown in SEQ ID NO: 2 , A polypeptide comprising an amino acid sequence deleted, inserted or added and having antibacterial activity without inhibiting protease The following nucleic acid (a) or (b):
(A) a nucleic acid consisting of the 499th to 648th base sequence of the base sequence shown in SEQ ID NO: 1 (b) a nucleic acid consisting of a sequence complementary to the 499th to 648th base sequence of the base sequence shown to SEQ ID NO: 1 Nucleic acid that hybridizes under stringent conditions and encodes a polypeptide having antibacterial activity without inhibiting protease The following polypeptide (a) or (b).
(A) Polypeptide consisting of amino acids 131 to 243 of the amino acid sequence shown in SEQ ID NO: 2 (b) Substitution of one or several amino acids in the amino acid sequence 131 to 243 of the amino acid sequence shown in SEQ ID NO: 2 , A polypeptide comprising an amino acid sequence deleted, inserted or added and having antibacterial activity without inhibiting protease A nucleic acid encoding the following polypeptide (a) or (b).
(A) Polypeptide consisting of amino acids 131 to 243 of the amino acid sequence shown in SEQ ID NO: 2 (b) Substitution of one or several amino acids in the amino acid sequence 131 to 243 of the amino acid sequence shown in SEQ ID NO: 2 , A polypeptide comprising an amino acid sequence deleted, inserted or added and having antibacterial activity without inhibiting protease The following nucleic acid (a) or (b):
(A) a nucleic acid comprising the nucleotide sequence 445-783 of the nucleotide sequence represented by SEQ ID NO: 1 (b) a nucleic acid comprising a sequence complementary to the nucleotide sequence 445-783 of the nucleotide sequence represented by SEQ ID NO: 1. Nucleic acid that hybridizes under stringent conditions and encodes a polypeptide having antibacterial activity without inhibiting protease Look including the nucleic acid of claim 2, 3, 5 or 6, wherein, and recombinant vectors expressing claim 1 or 4, wherein the polypeptide. A transformant transformed with the nucleic acid according to claim 2, 3, 5 or 6, or the recombinant vector according to claim 7, and expressing the polypeptide according to claim 1 or 4 .   The transformant according to claim 8, which is a plant.   The transformant according to claim 9, wherein the plant is a plant, a plant organ, a plant tissue, or a plant cultured cell.   The transformant according to claim 9 or 10, wherein the plant is a plant belonging to the family Gramineae.   The transformant according to claim 11, wherein the plant belonging to the family Gramineae is wheat.   The transformant of any one of Claims 9-12 which shows resistance with respect to a snow rot fungus.   The transformant according to claim 13, wherein the snow rot fungus is a red snow rot fungus.   The transformant according to any one of claims 9 to 12, which exhibits resistance to abiotic stress selected from the group consisting of low temperature, salt and dryness. A seed obtained from the transformant according to any one of claims 9 to 15, see containing nucleic acid of claim 2, 3, 5 or 6, wherein or a recombinant vector according to claim 7, And the seed which expresses the polypeptide of Claim 1 or 4 .   A method for producing a polypeptide having antibacterial activity without inhibiting protease, comprising culturing the transformant according to claim 8 and collecting the polypeptide from the obtained culture. A step of transforming a plant with the nucleic acid according to claim 2, 3, 5 or 6, or the recombinant vector according to claim 7 , and from a transformant expressing the polypeptide according to claim 1 or 4 to a plant A method for producing a resistant plant, comprising a step of regenerating a body.   19. The method of claim 18, wherein a plant resistant to snow rot fungi is produced.   19. The method of claim 18, wherein the plant is resistant to abiotic stress selected from the group consisting of low temperature, salt and dryness.   The method according to any one of claims 18 to 20, wherein the plant is a plant belonging to the family Gramineae.   The method according to claim 21, wherein the plant belonging to the family Gramineae is wheat. The following antimicrobial agent characterized by comprising at least one of (a) ~ (d).
(A) the polypeptide according to claim 1 or 4, (b) the nucleic acid according to claim 2, 3, 5 or 6, (c) the recombinant vector according to claim 7, (d) the transformant according to claim 8.   The antibacterial agent according to claim 23, which is used for a plant.   The antibacterial agent according to claim 23 or 24, which has antibacterial activity against snow rot fungi.

Images