JP3357915B2 - Gene cloning method using signal sequence - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for cloning a novel gene using a signal sequence. More specifically, the present invention relates to a method for cloning a novel gene encoding a protein localized in an organelle using a polynucleotide having a nucleotide sequence encoding a signal sequence related to localization in the organelle. Furthermore, the present invention relates to a subunit constituting a protein complex that functions in the respiratory chain of rice obtained by the cloning and a gene encoding the same. More specifically, the present invention relates to a rice COXVb protein and a coxVb gene encoding the same . Further, the present invention relates to a nucleotide sequence complementary to the coxVb gene, a method for suppressing the expression of the COXVb protein using the complementary nucleotide sequence, a so-called “antisense method”, and an antibody that binds to the COXVb protein. Furthermore, the present invention relates to a method for growing a new plant variety based on the use of a variant of the COXVb protein and coxVb gene.

[0002]

2. Description of the Related Art Mitochondria are organelles found in most eukaryotic cells and contribute to intracellular metabolism. The main role of mitochondria is as an energy conversion organ.
Combining P. Many enzymes function in ATP synthesis. For example, those that produce acetyl-CoA from pyruvate and fatty acids, acetyl-C by the citric acid cycle
Some substances oxidize oA. The final product produced in this oxidation process is CO ₂ and NADH, CO ₂ is discharged as waste from the cells, NADH is the main source of electrons flowing along the respiratory chain. The enzymes that make up the respiratory chain form a complex and are embedded inside mitochondria, and are called membrane-bound respiratory enzyme complexes. These enzyme complexes normally form an electrochemical proton gradient through which electrons move and eventually reach oxygen. To date, three major membrane-bound respiratory enzyme complexes have been identified and purified, called the NADH dehydrogenase complex, the cytochrome b-c1 complex, and the cytochrome oxidase complex, respectively. In the respiratory chain, the NADH dehydrogenase complex accepts electrons from NADH and passes them to ubiquinone via flavin and at least five iron-sulfur centers. Ubiquinone passes electrons to the next cytochrome b-c1 complex. The b-c1 complex then passes the received electrons to cytochrome c,
Is passed to the cytochrome oxidase complex, and finally from the cytochrome oxidase complex to oxygen. The most advanced of these membrane-bound respiratory enzyme complexes is the cytochrome oxidase complex, and in mammals,
It has been isolated as a dimer of about 300,000 daltons. The monomer is composed of nine or more polypeptides, and is known to contain two cytochromes and two copper atoms, and several genes encoding these polypeptides have been cloned. Despite such studies in mammals,
In plants, elucidation of the factors constituting the cytochrome oxidase complex and the mechanism of the respiratory chain has not been advanced.

[0003] Elucidation of the mechanism of mitochondrial respiratory chain, which is a place of energy conversion occupying an important position in the vital activities of cells, requires elucidation of the physiological functions of eukaryotic cells and living organisms and their regulation, and furthermore, molecular biology Is important for the creation of new varieties of plants by a statistical approach.

[0004] Elucidation of the function of organelles in eukaryotes and the like, not only in mitochondria, is also important for production of useful substances, creation of new plants, treatment of diseases, and the like. To that end, it is extremely important to elucidate the functions of organelle proteins, especially proteins localized in organelles.

[0005] In organelles such as mitochondria, cloning of a structural gene encoding a locally existing protein requires a great deal of effort in protein purification, amino acid sequencing, screening of a cDNA library, and nucleotide sequencing. Is done. This hinders the progress of gene and organelle function analysis. From homology search to polymerase chain reaction (PC
R) -based cloning methods have been developed,
These methods are limited to cloning of a gene having a high homology in base sequence to a known structural gene. Therefore, it is difficult for conventional techniques to efficiently clone a wide variety of unknown genes related to specific organelles having different nucleotide sequences and different functions of proteins. Therefore, a method for efficiently cloning a gene encoding a protein localized in a specific organelle has been desired.

[0006]

SUMMARY OF THE INVENTION The present invention has been made to solve the above problems, and has as its object the following:
An object of the present invention is to provide a novel gene isolation technique using a signal sequence contained in a precursor polypeptide. A method for cloning such a gene is provided, and the structure and function of the novel gene are clarified, thereby elucidating the structure and function of the organelle. Thus, the present invention opens the way to, for example, the cultivation of new varieties in plants or the creation of biosensors and artificial mitochondria.

It is another object of the present invention to provide a coxVb gene which encodes a subunit constituting a cytochrome oxidase complex which functions in electron transfer in a respiratory chain of a plant mitochondria, and further provides various variants of the coxVb gene. And a new plant variety using a polynucleotide having a sequence complementary to the coxVb gene.

[0008]

SUMMARY OF THE INVENTION The present invention relates to a method for cloning a gene encoding a protein localized in an organelle, comprising a polynucleotide having a nucleotide sequence encoding a signal sequence related to localization in an organelle. The present invention relates to a method comprising a step of performing hybridization using nucleotides as probes.

[0009] In a preferred embodiment, the organelle is mitochondria and the probe is a polynucleotide having a nucleotide sequence encoding a signal sequence associated with mitochondrial localization.

In a preferred embodiment, the probe has a nucleotide sequence that is a polynucleotide containing at least 15 contiguous nucleotides and is present in the nucleotide sequence shown by SEQ ID NO: 4 or 5.

In a preferred embodiment, the probe is a polynucleotide containing at least 15 contiguous nucleotides and has the nucleotide sequence shown by SEQ ID NO: 4 or 5.

In a preferred embodiment, the probe has a nucleotide sequence that encodes the amino acid sequence represented by SEQ ID NO: 6 or 7, which is a polynucleotide containing at least 15 contiguous nucleotides.

[0013] In a preferred embodiment, the probe is a polynucleotide containing at least 15 contiguous nucleotides, wherein one or more amino acids are added or deleted in the amino acid sequence represented by SEQ ID NO: 6 or 7. A polynucleotide encoding a lost or substituted amino acid sequence.

In a preferred embodiment, the probe is a polynucleotide containing at least 15 contiguous nucleotides and has a nucleotide sequence encoding the amino acid sequence shown by SEQ ID NO: 8 or 9.

[0015] In a preferred embodiment, the probe is a polynucleotide containing at least 15 contiguous nucleotides, wherein one or more amino acids are added or deleted in the amino acid sequence represented by SEQ ID NO: 8 or 9. A polynucleotide encoding a lost or substituted amino acid sequence.

The present invention relates to a rice COXVb protein, wherein the polypeptide sequence is an amino acid sequence represented by SEQ ID NO: 2, and an amino acid sequence in which one or more amino acids are added, deleted or substituted in the amino acid sequence. A protein having a biologically equivalent activity to said COXVb or a fragment thereof.

The present invention provides a polynucleotide having a coxVb gene encoding rice COXVb, wherein the nucleotide sequence of the gene is an amino acid sequence represented by SEQ ID NO: 2, and one or more amino acids are added to the amino acid sequence. The present invention relates to a polynucleotide which is a deleted or substituted amino acid sequence which is a sequence encoding a biologically equivalent activity of COXVb or a fragment thereof.

The present invention also relates to the aforementioned, further comprising an intron.
Regarding the coxVb gene.

The present invention relates to a recombinant vector having the coxVb gene.

The present invention provides a method for producing the COXVb protein, which comprises culturing cells having a vector containing the gene under appropriate conditions under which the gene encoding the protein is expressed, and And recovering the protein produced in step (a).

The present invention relates to a polynucleotide sequence having a sequence complementary to all or part of the nucleotide sequence of the coxVb gene or a sequence sufficiently complementary to form a double strand with the nucleotide sequence of the gene.

The present invention relates to a vector having a sequence complementary to the above-mentioned polynucleotide sequence and being connected to a promoter so as to produce a polynucleotide complementary to the above-mentioned nucleotide sequence.

The present invention relates to a method for suppressing the function of the coxVb gene in a cell, comprising the step of introducing the vector into the cell.

[0024] The present invention relates to the above-mentioned polypeptide,
Related to antibodies.

[0025] In a preferred embodiment, the antibody is
It is a polyclonal antibody.

In a preferred embodiment, the antibody is
It is a monoclonal antibody.

[0027] The present invention relates to a plant cell having a nucleotide sequence obtained by modifying the coxVb gene.

[0028]

DETAILED DESCRIPTION OF THE INVENTION The present invention, unless otherwise indicated,
Molecular biology, biochemistry, genetics, genetic engineering, microbiology,
And conventional techniques in fields such as plant breeding.

As used herein, the term "polynucleotide" refers to a polymer of nucleotides, and is not limited to a specific chain length.

As used herein, the term "vector" refers to a foreign polypeptide in which an exogenous polypeptide is operably linked to elements necessary for its transcription and translation, and which is necessary for replication in a host cell and selection of a recombinant. A medium for transmitting a foreign polypeptide into a host cell, comprising various factors. Preferred vectors are plasmids. Those that transmit to plant cells or yeast and / or bacteria are particularly preferred. It is well known in the art that the type of the above-described vector of the present invention and the type of each regulatory element used can be changed depending on the host cell.

As used herein, “organelle” refers to a structural unit having a certain function present in eukaryotes,
Mitochondria, peroxisomes, plastids, endoplasmic reticulum,
Includes Golgi apparatus, lysosomes, endosomes, and secretory vesicles.

As used herein, the term "library" refers to
It refers to a nucleic acid such as genomic DNA or cDNA containing the gene of interest linked to an appropriate vector such as a plasmid or phage. That the type of library can vary depending on the screening method used,
It is well known to those skilled in the art.

As used herein, the term "complementary sequence"
A sequence that encodes a ribonucleotide capable of forming a double-stranded ribonucleotide complex by hydrogen bonding with a transcript of a specific gene.

As used herein, "antibody or polyclonal antibody" includes proteins produced upon immunization with an antigen or receptor. The term "monoclonal antibody" includes immunoglobulins derived from a single clone of the cell. All monoclonal antibodies derived from this clone are chemically and structurally identical and specific for one antigenic determinant.

As used herein, the term "signal sequence"
Includes polypeptide sequences encoded on the chromosome that are not normally found in the mature protein, which are involved in the localization of the precursor protein encoded downstream of the sequence.

As used herein, the term “hybridization” refers to a single-stranded analyte polynucleotide and a single-stranded complementary to its sequence used in genetic research, biochemical research, and clinical diagnosis. A technique for hybridizing with a nucleic acid probe and detecting a signal released from a labeled double-stranded polynucleotide, including various modified methods. For example, Maniatis et al., Molecular Cloni
ng: A Laboratory Manual Cold Spring Harbor Press,
See Cold Spring Harbor, NY (1989).

In the present invention, a “probe” is a labeled polynucleotide capable of providing a detectable signal, and the labeling can be performed using fluorescence, radioactivity, color development, magnetism, enzymatic activity and the like. .

In the present invention, “modification of one or more amino acids” refers to a well-known technique of site-directed mutagenesis (for example, Nucleic Acids Res., 10: 6487-6500 (1982)).
Or the PCR method (see, for example, Saiki et al. (19)
88), Science, 230: 1350-1354).
It means a modification of an amino acid that can be added, deleted or substituted.

The present inventor has proposed a type of ribosomal protein
Rice encoding mitochondrial RPS11s11-1Before
From the results of amino acid sequence analysis of the precursor sequence,s11-1
Is a precursor arrangement of a plant-derived ATPase β subunit.
High homology with the N-terminus of the sequence; ands11-
1Polypeptide located at the N-terminus of RPS11 encoded by
Functions as a mitochondrial transport signal
Was found. For this reason, multiple functionally different tanks
Protein precursors share highly homologous signal sequences
It is suggested that The present inventor has proposed a specific signal distribution.
Column is the precursor of another gene due to gene duplication and recombination
We hypothesized that it functions as a body array.

To prove this hypothesis, the inventor
Plaque hybridization was performed using a polynucleotide at the 5 'end of another gene (another RPS11-encoding copy gene s11-2 present in the nuclear genome of rice) as a probe, and it was completely functional with ribosomal proteins. A novel gene, coxVb , encoding mitochondrial cytochrome c oxidase complex subunit Vb, which differs in structure and structure, was successfully isolated. Hereinafter, the present invention will be described in detail.

The present invention is based on the hypothesis that proteins having different functions share a signal sequence with high homology and are consequently localized to a specific organelle. First, Table 1 shows an amino acid sequence consisting of 35 amino acid residues at the N-terminal part of the precursor of ribosomal protein RPS11 encoded by the s11-1 gene (rice RPS11 in the table)
FIG. 4 compares the N-terminal partial amino acid sequences (46 to 55 amino acid residues) of three types of mitochondrial protein precursors encoded in the plant nuclear genome. NpBATP in the table
ase, HbBATPase and OsBATPase are each Nicotiana p
lumbaginifolia , Hevea brasiliensis and Oryzae sat
Figure 7 shows the mitochondrial ATPase β subunit from iva .

[0042]

[Table 1]

As shown in Table 1, the amino acid sequence of the N-terminal part of the RPS11 precursor encoded in the rice nuclear genome, which is a protein having different functions and origins, and the N-terminal part of the mitochondrial protein precursor of other plants Has a high homology with the amino acid sequence. This supports our hypothesis that proteins with different functions share a highly homologous signal sequence and are consequently localized to specific organelles.

Furthermore, the present inventor assumed that a signal sequence with high homology was conserved between other protein precursors, and that 5% of other copy gene s11-2 of RPS11 present in the rice genome was Plaque hybridization was performed using the putative signal sequence at the 'end. As a result, we succeeded in isolating a novel mitochondrial gene, coxVb , encoded in the nucleus. This proved that the inventor's hypothesis was correct.

Table 2 is a comparison table of the nucleotide sequences of the 5 'terminal region of the novel rice coxVb isolated by the method of the present invention and the s11-2 gene and the vicinity thereof. -In the table
Indicates a deletion, and * indicates that both nucleotide sequences are identical. The nucleotide sequence homology between rice coxVb and s11-2 is 86% identical over 145 nucleotides.
This indicates that a precursor sequence having high homology is conserved among a plurality of genes, and further supports the above hypothesis of the present inventors.

[0046]

[Table 2]

The fact that the novel gene isolated by the method of the present invention was rice coxVb was the result of amino acid sequence homology analysis.

Rice coxVb , rice s11-2 , cytochrome c oxidase complex subunit Vb encoded in the human genome
Table 3 shows a comparison of all amino acid sequences between (human COXVb) and cytochrome c oxidase complex subunit IV (yeast COXIV) encoded in the yeast genome.

[0049]

[Table 3]

Each amino acid residue is represented by one letter code. Rice COXVb, human COXVb, East COXIX, s1 in the table
1-2 are rice COXVb, human COXVb, yeast COXIV, rice s1, respectively.
1-2 represents a protein. -In the table indicates a deletion.

The amino acid sequence homology between rice COXVb and human COXVb or yeast COXIV is 49% and 35%, respectively. Based on the results of the amino acid sequence homology analysis, it was found that the novel gene isolated by the method of the present invention was a coxVb gene encoding a subunit Vb constituting a cytochrome oxidase complex in rice.

From this, it was proved that a novel gene which had not been found can be isolated by using the polynucleotide encoding the signal sequence of the present invention.

Based on the results of the above research, the present inventor carried out hybridization using a polypeptide having a nucleotide sequence encoding a signal sequence present in a specific protein precursor to obtain a homologous signal sequence. Finding that different genes can be isolated,
The present invention has been completed. According to the present invention, a novel gene encoding a protein localized in a specific organelle in a eukaryote can be isolated.

Hereinafter, the present invention will be described using mitochondria as an example of an organelle.

The cloning of a gene encoding a protein localized in the mitochondria of a plant is performed by cloning at least one gene located at the 5 ′ end of the s11-2 gene or the coxVb gene.
This is achieved by screening a plant-derived library using five consecutive polynucleotides as probes. Polynucleotides used as probes can be prepared from natural materials or can be synthesized by methods known in the art. For example, prepared using, but not limited to, polynucleotide cloning, chemical crosslinking techniques, chemical synthesis, enzymatic assembly, combinations thereof, or other suitable methods. The probe is a polynucleotide having a nucleotide sequence present in the nucleotide sequence represented by SEQ ID NO: 4, or the amino acid sequence represented by SEQ ID NO: 5, or one or more amino acids in the amino acid sequence represented by SEQ ID NO: 5 May be a polynucleotide having a nucleotide sequence encoding an added, deleted or substituted amino acid sequence. Here, "one or more amino acids" refers to a well-known site-directed mutagenesis method (for example, Nucleic Acids Res., 10: 6487-).
6500 (1982)) or the PCR method (eg,
(See Saiki et al. (1988), Science, 230: 1350-1354)) by amino acids that can be added, deleted or substituted. In addition, such probes may comprise a mixture of polynucleotides encoding the same amino acid but having different nucleotide sequences, a so-called "mixed probe" (see, eg, Maniatis et al., Supra). It can also be in the form.

The cloning of other novel genes is achieved by screening libraries from eukaryotes having organelles. Preferably, a mitochondrial DNA library, a cDNA library or a nuclear genomic DNA library is prepared by a conventional method using a λ phage cloning vector, and can be used for screening. Commercially available genomic libraries can also be suitably used.

Using a polynucleotide fragment encoding a signal sequence located at the 5 'end as a probe,
By performing plaque hybridization, a cDNA clone having a sequence homologous to the probe can be selected. The polynucleotide used for the probe encodes a naturally occurring nucleotide sequence encoding the amino acid sequence of a specific protein, or an amino acid sequence in which one or more amino acids have been added, deleted or substituted in the amino acid sequence of the protein. May have a nucleotide sequence. Hybridization can be performed under general conditions known to those skilled in the art. The degree of stringency can vary within a range known to those skilled in the art. For example, Maniat
See is et al., supra. From the obtained positive clones,
Obtain a DNA fragment from the library. Next, the nucleotide sequence of the DNA fragment can be determined by a nucleotide sequence analysis method known in the art (such as the dideoxy method of Sanger et al.) Or a commercially available automatic sequencer. The homology analysis is performed using the amino acid sequence deduced from the determined nucleotide sequence, and the protein encoded by the cloned gene is estimated. Expression of the cloned gene can be accomplished by constructing an expression vector compatible with a suitable prokaryotic or eukaryotic host into which the cloned DNA has been inserted, introducing the expression vector into a host cell, and culturing and producing the recombinant host cell. Is achieved by the recovery of The function of the protein is determined by methods known in the art,
For example, it can be identified by creating a deletion strain or an overexpression strain of the gene encoding this protein and analyzing their phenotype.

Hereinafter, the cloning of the coxVb gene and its expression will be described. It goes without saying that these methods are used for screening and expression of other proteins of other organelles or mitochondria.

Using the polynucleotide obtained above, which encodes a putative signal sequence located at the 5 ′ end of the s11-2 gene, as a probe, hybridization to a rice-derived library is performed. Can select a clone containing the coxVb gene. The rice-derived library to be used is prepared by a conventional method using a λ phage cloning vector, and a mitochondrial DNA library, a cDNA library, or a nuclear genomic DNA library can be used for screening. Commercially available genomic libraries can also be suitably used. Hybridization can be performed using various methods known to those skilled in the art. Plaque hybridization may be suitably used, but is not limited thereto. Hybridization conditions can be performed under conditions known to those skilled in the art. See, for example, Maniatis et al., Supra. A positive clone obtained by hybridization can be isolated, DNA can be extracted, and the nucleotide sequence can be determined. The determination of the nucleotide sequence can be achieved by a general nucleotide sequencing method. The obtained nucleotide sequence and deduced amino acid sequence are subjected to homology analysis by a homology analysis program through a generally available database to identify the coxVb gene. This coxVb gene can be used to carry out the method of the present invention.

The cloning of the coxVb gene of the present invention can be performed using a rice-derived library in the same manner as described above. Alternatively, a clone containing the coxVb gene present in the plant nuclear genome can be isolated by directly using a part of the nucleotide sequence of the coxVb gene represented by SEQ ID NO: 1 as a probe.

Furthermore, using the rice genomic DNA or cDNA contained in the above library as a template, PCR (polymerase chain reaction) (Saiki et al. (1988), Science, 230:
1350-1354), and it is easy to use the amplified nucleotide sequence containing the coxVb gene. Alternatively, a DNA fragment chemically synthesized by a known DNA synthesis method (for example, a phosphoramidite method) based on the nucleotide sequence information disclosed in the present specification is also suitably used as the coxVb gene of the present invention. obtain.

In the production of the coxVb gene of the present invention, a small change in the primary structure of the naturally occurring amino acid sequence of COXVb represented by SEQ ID NO: 3 is caused by the subunit constituting the cytochrome oxidase complex of the present protein. It can be understood by those skilled in the art that the operation can be performed within a range that does not lose the function of. Such changes include, within the amino acid sequence of the present signal peptide,
The addition, deletion or substitution of one or more amino acids is included, and site-specific mutagenesis (site
-directed mutagenesis) (for example, Nucleic Acids Re
s., 10: 6487-6500 (1982)) or the PCR method (Saiki et al. (1988), Science, 230: 1350-1354). Here, “one or more amino acids”
It means the number of amino acids that can be added, deleted or substituted by the site-directed mutagenesis method or the PCR method.

The obtained gene of the present invention can be used as it is or by connecting an appropriate linker to construct a recombinant vector shown below.

The coxVb gene and various regulatory elements for expressing the gene in a host cell (including a promoter, a ribosome binding site, a terminator, an enhancer, and various cis elements for controlling the expression level) are included. In the construction of a recombinant expression vector, a technique of restriction of polynucleotides by various restriction enzymes and a technique of ligation of polynucleotide fragments, which are well understood in the art, are used. The type of regulatory element used in the vector and its construction will depend on the type of host cell.

When a yeast is used as a host, a preferred vector is a YEp type vector having a 2μ plasmid replication system (ori), but is not limited thereto, and other expression vectors may be used. The use of yeast and E. coli shuttle vectors is preferred. As a promoter to be used, yeast F1-
ATPase, a promoter for dihydrofolate reductase, or a promoter for an enzyme in various glycolysis systems, such as a promoter for alcohol dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase phosphoglycoisomerase, etc., can be preferably used. .

These promoters are used together with regulatory sequences such as enhancers and operators. As the reporter gene, a yeast F1-ATPase gene and a dihydrofolate reductase gene can be suitably used, but are not limited thereto. As a yeast host, common Saccharo
In addition to myces cerevisiae , Zygosaccharomyces rouxii , Sc
hizosaccharomyces pombe , as well as Pichia and Candida
Etc. can also be used. The use of an amino acid auxotrophic or base auxotrophic strain is preferred.

An example of construction of the above-described vector when a plant cell is used as a host will be described below. As the promoter, a cauliflower mosaic virus (CaMV) 35S promoter, a nopaline synthase (NOS) promoter and the like can be generally used, but are not limited thereto. The terminator that can be used in the practice of the present invention is not particularly limited. The terminator region of CaMV35S or NOS can be suitably used. A plasmid (for example, a pBI-type plasmid (Clontech) which can be used to introduce the produced gene construct into a plant via Agrobacterium genus bacteria.
)) Or a pUC plasmid, the recombinant vector of the present invention using a plant cell as a host can be constructed.

The recombinant vector of the present invention is
In addition to the leotide construct, additional selectable marker inheritance
May contain offspring. The selectable marker gene can be used in the present invention.
To select host cells into which the polynucleotide has been introduced.
Used for Selectable markers that can be commonly used
-The gene depends on the host cell used. For example,
In the case of yeast, genes that complement auxotrophy (Leu,Ur
a,TrpKanamycin resistance is added for plant cells.
Neomycin phosphotransferase II (NTPI
I) or hygromycin conferring hygromycin resistance
Antibiotic resistance such as isin phosphotransferase (HPT)
Sex genes may be suitably used, but are not limited thereto.
No.

Depending on the host cell used, transformation of the host cell with the above vectors may be performed using standard techniques appropriate for that cell.

For prokaryotic cells, Cohen, SN et al. (197
2) (Proc. Natl. Acad. Sci. USA, 69, 2110) The calcium chloride treatment method reported can be used. Electroporation can be applied to eukaryotic cells such as yeast and plant cells in addition to prokaryotic cells.

In certain higher plant cells, Agrobacterium tumefaciens (Agrobacterium tum
efaciens) (Shaw, CH et al. (1983), Gen.
e, 23: 315) can be widely used. In this method, first, a constructed vector (pBI plasmid or the like) is introduced into Agrobacterium tumefaciens by electroporation or the like, and then the transformed bacteria are subjected to a leaf disk method (Horsch, RB et al.). (1985), Scien
ce, 227: 1229-1231) and the like, and introduced into host plant cells.

Production of a target protein by a host cell that has acquired the COXVb protein-producing ability by introducing a vector can be achieved by culturing the above-mentioned host cell under appropriate conditions such that COXVb is expressed. The appropriate conditions are
It can be determined using methods well known to those skilled in the art. See, for example, Maniatis et al., Supra, incorporated herein by reference. The host cells are separated by centrifuging or filtering the culture to obtain a supernatant. Precipitate the protein from the supernatant by ordinary means, for example, salting out, solvent precipitation (eg, ethanol, acetone, etc.), or
The fraction containing the target polypeptide is obtained by concentration by ultrafiltration (for example, Diaflow membrane YC, manufactured by Amicon). This fraction is further subjected to precipitation, filtration, centrifugation, desalting and other treatments to obtain a crude polypeptide. The purified polypeptide can be recovered by appropriately combining ordinary polypeptide purification means such as lyophilization, isoelectric point precipitation, electrophoresis, ion exchange chromatography, and crystallization.

Further, the present invention relates to coxVb encoding COXVb.
A polynucleotide having a sequence complementary to the nucleotide sequence of a gene, a vector capable of producing a polynucleotide having the complementary sequence, and a method for suppressing the function of a COXVb protein including a step of introducing the vector into cells, It relates to the so-called “antisense method”. co
Genetically modified plants, fungi, or yeast that reduce ATP production by suppressing the expression of the xVb gene, resulting in pollen degeneration (male sterility) and flower mutation due to an imbalance between energy use and production Can be created.
A polynucleotide having a sequence complementary to the nucleotide sequence encoding COXVb was obtained as described above.
It is available as a complementary strand of the gene encoding COXVb. Therefore, a vector capable of producing a polynucleotide having a complementary sequence has a polynucleotide encoding COXVb in the opposite direction (antisense) to the direction in which COXVb is originally expressed in an appropriate vector as described above. Can be constructed by Details of the type of vector and the regulatory element used for its construction and the method of construction are the same as described above.

The method of suppressing the function of the coxVb gene can be achieved by introducing a vector capable of producing a polynucleotide sequence having a sequence complementary to the nucleotide sequence encoding COXVb into a host cell. The details of the process of introducing the vector into the host cell are the same as described above.

Further, the present invention provides an antibody capable of specifically forming a complex with the COXVb protein. Such an antibody is useful as a marker when fractionating rice mitochondria into inner membrane, outer membrane, matrix and the like.
Methods for producing polyclonal and monoclonal antibodies are well-known in the art. Harlow and Lane, An, incorporated herein by reference.
tibodies: A Laboratory Manual, Cold Spring arbor
See Laboratory, New York (1988). Production of the monoclonal antibodies of the present invention can be performed biologically by introducing COXVb or a fragment thereof into an animal, for example, a mouse or rabbit. Briefly, the process involves injecting the immunogen into a mouse, or other suitable animal. Subsequently, the animal is sacrificed and cells from the spleen are fused with myeloma cells. As a result, hybrid cells called “hybridomas” can be obtained. The hybridoma population is screened and individual clones are isolated. Each clone secretes a single antibody species against the antigen. After screening the different hybridoma cell lines to identify those that produce antibodies to the desired antigen, the antibodies produced by the individual hybridoma cell lines are preferably screened to have the highest affinity for COXVb. Identify antibodies with specificity.

The present invention further relates to a method for creating a new plant variety by preparing cells having a genome containing various variants of the coxVb gene. Introduction of a mutation into the coxVb gene can increase or decrease the activity of cytochrome oxidase. Mutation of the mutation can be performed by a method known in the art, point mutagenesis, random mutagenesis,
Methods include, but are not limited to, methods of introducing insertions and deletions. For example, Kunkel, TA Proc.Natl.Acad.
See Sci. USA 82, 488 (1985) and Maniatis, T et al., Supra. In plants with increased activity, high productivity of ATP can be created due to high production of ATP.

The present invention will be described in more detail in the following examples, which do not limit the present invention in any way.

[0078]

EXAMPLES Example 1 (Isolation of coxVb gene from rice nuclear genome) (1). Preparation of genomic library 100 g of mature rice leaves were mixed with 1 liter of a homogenization buffer (0.44 M saccharose, 2.5% ( w / v) Ficoll-400 (Pha
rmacia), 5% Dextran 40, 25 mM Hepes (pH 7.5),
10 mM MgCl ₂ , 10 mM β-mercaptoethanol and 0.1 mM
Homogenized in 1% (w / v) polyvinylpyrrolidone). This homogenate was filtered and centrifuged at 3,000 g for 5 minutes. The precipitate is then resuspended in 50 ml of the above homogenization buffer containing 0.5% (w / v) Triton X-100,
Centrifuged at 3,000 g for 5 minutes. 20 ml of the resulting precipitate
50 mM Tris-HCl buffer (containing 10 mM EDTA, 0.4 mg / ml proteinase K, and 2% (w / v) sarkosyl, pH 7.
5) dissolved in. Insolubles were removed by centrifugation at 20,000 g for 5 minutes. The resulting DNA was purified by cesium chloride density gradient centrifugation in the presence of ethidium bromide. High molecular weight nuclear genomic DNA was digested with a restriction enzyme Sau3A, and the obtained approximately 15 to 20 kb fragment was separated and purified by sucrose density gradient centrifugation. These fragments were inserted into the BamHI site of the λ phage vector DASHII (Stratagene),
A rice genome library was prepared.

(2). Preparation of cDNA Library Total RNA was extracted from mature leaves of rice and poly-A-added RNA was prepared according to a conventional method (Ausubel, FM et al., “Curre
nt Protocols in Molecular Biology ”(John Wiley & S
ons, Inc., NY, 1987)). Pharmacia c
Using a DNA synthesis kit, cDNA synthesized from the poly-A-added RNA was converted to a lambda ZAPII vector (Stratage
ne) (EcoRI site) to prepare a rice cDNA library.

[0080] (3). Using as a probe XhoI-NotI fragment of about 0.8kb isolated rice coxVb gene rice s11-2 genes each library screening and positive clones. Multi-labeling system (Amersham,
Buckinghamshire, UK) using [α- ³² P] d
Labeled with CTP. Conventional plaque hybridization methods using labeled probes (Sambrook et al., 198
According to 9), the libraries prepared in (1) and (2) above were screened, and positive genomic clones and cDNA clones hybridized to the nucleotide sequence encoding the signal sequence of the s11-2 gene were isolated. .

Example 2 ( Sequencing of coxVb gene) The above isolated positive clone was subcloned into the vector pBluescriptIISK- (Stratagene). The obtained subclones were subjected to a normal deletion operation using exonuclease III (Henikoff, S. (1984), Gene, 28: 351) to prepare a series of deleted DNA fragments. Obtained series of deleted DNA
Using the fragment, the nucleotide sequence of the positive clone was determined using a commercially available DNA sequencer (ALF DNA Seaquencer II, manufactured by Pharmacia) according to the instructions provided by the manufacturer. As shown in SEQ ID NO: 1, this clone was found to have an open reading frame ( coxVb ) of 507 bp across five introns.

Example 3 (Computer analysis of coxVb sequences) Nucleotide and putative amino acid sequences were obtained using computer software GENETYX (Software Development Co., LTD., Tokyo, Japan).
Japan). Analysis of nucleotide and amino acid sequence homology was performed through the DDBJ / EMBL / GeneBank database using the FASTA program (Pearson and Lipman,
1988) and BLAST (Altschul et al., 1990). Table 3 shows a comparison of all amino acid sequences of the above COXVb with the cytochrome c oxidase subunit Vb (COXVb) encoded in the human genome and the cytochrome c oxidase complex subunit IV (COXIV) encoded in the yeast genome. It is a table. Each amino acid residue is represented by one letter code.
-In the table indicates a deletion. Amino acids shown in white are CO
XVb is identical to the amino acid residue at that position. The amino acid sequence homology between COXVb and COXVb or COXIV is 49% and 35%, respectively.

The present inventor has determined that the present COXVb encodes a subunit Vb constituting a cytochrome oxidase complex in rice based on the results of the above amino acid sequence homology analysis.
coxVb gene.

[0084]

According to the present invention, there is provided a method for isolating a gene derived from a nuclear genome encoding a protein localized in an organelle. Further, a coxVb gene encoding a subunit constituting a cytochrome oxidase complex that functions in the respiratory chain of plant mitochondria is provided, and further, various variants of the coxVb gene or coxVb
A new plant variety having a sequence complementary to the gene is provided.

[0085]

[Sequence List] SEQ ID NO: 1 Sequence length: 507 Sequence type: Nucleic acid Number of strands: Double-stranded Sequence type: cDNA to mRNA sequence ATGTGGCGCC GCCTCCAAAC CCTGGCCCCG GCCCTGCGCC GCGCCGCCTC CGCCTCCGCC 60 TCCGCGGCGG CATCGGGCGC CGCGGGCCCC GCCTCCCGCC GCCGCGCGCGCC CCGCCGCACC AGCCCTCTCC TGTCCGGGGA CAAGCCGGCG 180 ACGGTGGAGG ACGTCATGCC CATCGCCACG GGGCTGGAGC GCGAGGAGCT CGCGGCGGAG 240 CTCAAGGGGG AGAAGCGGTT CGACATGGAT CCTCCCGTAG GCCCATTTGG TACCAAGGAG 300 GCCCCAGCTG TCATTGAATC CTACTATAAC AAGAGGATAG TTGGTTGTCC TGGTGGTGAA 360 GGAGAGGATG AGCATGATGT TGTATGGTTC TGGTTGAAAA AAGATGAACC GCATGAGTGC 420 CCAGTCTGTT CACAATATTT TGTGCTTAAG GTCATTGGTG ATGGTGGTGA TCCAGATGGA 480 CATGATGATG ACGACGAGCA TCACCAC 507 SEQ ID NO: 2 sequence of Length: 169 Sequence type: Amino acid Sequence type: Protein Sequence Met Trp Arg Arg Leu Gln Thr Leu Ala Pro Ala Leu Arg Arg Ala Ala 1 5 10 15 Ser Ala Ser Ala Ser Ala Ala Ala Ser Gly Ala Ala Gly Pro Ala Ser 20 25 30 Leu Ala Arg Ala Ala Pro Leu Ser Thr Ala Ala Ala Ala Ala Phe Arg 35 40 45 Arg Thr Ser Pro Leu Leu Ser Gly Asp Lys Pro Ala Thr Val Glu Asp 50 55 60 Val Met Pro Ile Ala Thr Gly Leu Glu Arg Glu Glu Leu Ala Ala Glu 65 70 75 80 Leu Lys Gly Glu Lys Arg Phe Asp Met Asp Pro Pro Val Gly Pro Phe 85 90 95 Gly Thr Lys Glu Ala Pro Ala Val Ile Glu Ser Tyr Tyr Asn Lys Arg 100 105 110 Ile Val Gly Cys Pro Gly Gly Glu Gly Glu Asp Glu His Asp Val Val 115 120 125 Trp Phe Trp Leu Lys Lys Asp Glu Pro His Glu Cys Pro Val Cys Ser 130 135 140 Gln Tyr Phe Val Leu Lys Val Ile Gly Asp Gly Gly Asp Pro Asp Gly 145 150 155 160 His Asp Asp Asp Asp Glu His His His 165 SEQ ID NO: 3 Sequence length: 59 Sequence type: Amino acid Sequence type: Protein Sequence Met Trp Arg Arg Leu Gln Thr Leu Ala Pro Ala Leu Arg Arg Ala Ala 15 10 15 Ser Ala Ser Ala Ser Ala Ala Ala Ser Gly Ala Ala Gly Pro Ala Ser 20 25 30 Leu Ala Arg Ala Ala Pro Leu Ser Thr Ala Ala Ala Ala Ala Phe Arg 35 40 45 Arg Thr Ser Pro Leu Leu Ser Gly Asp Lys Pro 50 55 SEQ ID NO: 4 Sequence length: 268 Sequence type: Nucleic acid Number of strands: Double-stranded Sequence type: genomic DNA sequence ATCAAATCGA GAGAGAGCCC CCATCTCGCC TCGCCTCGCC GCCTCCTCCC TCCCCCCATT 60 CCCCCGCCGC CGCCGCTGCG AGCGCCCGGAGCTGCCGCCGCCCGCCGCGACCTGCCGCCGCCCGCT GCCTCCGCCT CCGCCGCGGG 180 CCCCGCCTCC CTCGCCAGCG CCGCGCCGCT CTCCACGGCG GCCGCCGCCG CCTTCCCACC 240 TCCTCAGTCT AAAACCGCTT CACGTATG 268 SEQ ID NO: 5 Sequence length: 287 Sequence type: Nucleic acid Number of strands: Number of double strands Sequence type: genomic DNA sequence GCCGCCGCCG CCGAGGGCGC CGCGACCTGA GAGCACACGC ACGCCCGCCA TGTGGCGCCG 120 CCTCCAAACC CTGGCCCCGG CCCTGCGCCG CGCCGCCTCC GCCTCCGCCT CCGCGGCGGC 180 ATCGGGCGCC GCGGGCCCCG CCTCCCTCGC CCGCGCCGCG CCGCTCTCCA CGGCGGCCGC 240 CGCCGCCTTC CGCCGCACCA GCCCTCTCCT GTCCGGGGAC AAGCCGG 287 SEQ ID NO: 6 SEQ length: 59 SEQ type: amino acid sequence of type: Tampa Met Arg Ala Arg Leu Gln Thr Leu Ala Pro Ala Leu Arg Arg Ala Ala 1 5 10 15 Ser Ala Ser Ala Thr Gly Pro Ala Ser Ala Ser Ala Ala Gly Pro Ala 20 25 30 Ser Leu Ala Ser Ala Ala Pro Leu Ser Thr Ala Ala Ala Ala Ala Phe 35 40 45 Pro Pro Pro Gln Ser Lys Thr Ala Ser Arg Met 50 55 SEQ ID NO: 7 Sequence length: 59 Sequence type: Amino acid Sequence type: Protein Sequence Met Trp Arg Arg Leu Gln Thr Leu Ala Pro Ala Leu Arg Arg Ala Ala 1 5 10 15 Ser Ala Ser Ala Ser Ala Ala Ala Ser Gly Ala Ala Gly Pro Ala Ser 20 25 30 Leu Ala Arg Ala Ala Pro Leu Ser Thr Ala Ala Ala Ala Ala Phe Arg 35 40 45 Arg Thr Ser Pro Leu Leu Ser Gly Asp Lys Pro 50 55 SEQ ID NO: 8 Sequence length: 20 Sequence type: Amino acid Sequence type: Protein Sequence Met Trp Arg Arg Leu Gln Thr Leu Ala Pro Ala Leu Arg Arg Ala Ala 1 5 10 15 Ser Ala Ser Ala 20 SEQ ID NO: 9 Sequence length: 21 Sequence type: amino acid Sequence type: Protein Sequence Ala Ala Gly Pro Ala Ser Leu Ala Arg Ala Ala Pro Leu Ser Thr Ala 1 5 10 15 Ala Ala Ala Ala Phe 20

──────────────────────────────────────────────────続 き Continued on the front page (58) Fields surveyed (Int. Cl. ⁷ , DB name) C12Q 1/68 C12N 15/00-15/09 BIOSIS (DIALOG) GenBank / EMBL / DDBJ / GeneSeq

Claims (2)

(57) [Claims] 1. A method for cloning a gene encoding a protein localized in plant chloroplasts, plant leaves
Signa, a gene encoding a protein located in the chloroplast
A hybridization using a polynucleotide having a nucleotide sequence encoding a nucleic acid sequence as a probe. 2. The method according to claim 2, wherein said nucleotide sequence is at least 15
Pieces of a continuous to contain nucleotide Runu Kureochido sequence <br/>, The method of claim 1.