JP2001327288A - Plastid sigma factor-binding protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for enhancing the expression of a protein in a chloroplast. SOLUTION: The subject protein is (a) a polypeptide having a specific sequence, or (b) a polypeptide that comprises a polypeptide modified by substituting, deleting or adding one amino acid or more in, from or to polypeptide (a) and has a plastid σ factor-binding activity.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a plastid sigma factor binding protein, a gene encoding the protein, a chloroplast genome, a source of a transformed plant, and a method for expressing a heterologous protein in chloroplasts.

[0002]

2. Description of the Related Art Chloroplasts are abundant in plant leaves and are organelles involved in photosynthesis, and are contained in plant cells in an amount of one to several hundreds.

[0003] The chloroplast genome is derived from mosses, tobacco,
The entire structure has been determined in rice, pine, Arabidopsis and the like, and the genome is divided into several types, but has been found to be universal in plants.

Furthermore, since the chloroplast genome is of a prokaryotic type and has homology to Escherichia coli genes, it is useful to elucidate the mechanism of controlling protein synthesis in chloroplasts, especially the mechanism of increasing protein expression. The protein can be produced in a large amount, and is expected to be applied not only to higher plants and algae, but also to prokaryotes such as cyanobacteria.

An object of the present invention is to provide a technique for increasing the expression of a useful protein in chloroplasts.

[0006]

SUMMARY OF THE INVENTION When excess light hits a plant, D1 forms a reaction center complex of chloroplast photosystem II.
And D2 protein are known to be damaged, but the mechanism of avoiding the effects of the damage caused by the light has not been sufficiently clarified.

The present inventors have noticed that when the D1 and D2 proteins are damaged in the presence of excessive light, the transcription of genes encoding these proteins is promoted. As a result of searching for factors, a novel protein shown in SEQ ID NO: 2 was unexpectedly produced by light stimulation, and the protein was converted to RNA polymerase (Plas
tid-encoded plastid RNA polymerase (PEP)
The affinity of plastid sigma factor, ie, PEP, for the promoters of the D1 and D2 proteins, particularly the D2 protein gene, is dramatically increased, and the expression of the D2 protein gene is increased by 6000.
It was found that the enhancement was about twice.

The present invention relates to a polypeptide of the following (a) or (b): (a) a polypeptide represented by SEQ ID NO: 2; (b) one or more amino acids in the polypeptide (a) are substituted. And polypeptides having a plastidic sigma factor binding activity.

Further, the present invention provides the following (a) and (b)
A gene consisting of any of the following polynucleotides: (a) a base sequence represented by SEQ ID NO: 1 or a complementary strand thereof, and (b) a polynucleotide consisting of the above base sequence (a) hybridized under stringent conditions. A soybean polynucleotide.

Further, the present invention provides a chloroplast genome having an expression cassette containing a gene encoding a useful polypeptide downstream of a promoter of the D1 or D2 protein gene or an analog thereof; a source of a transformed plant having the genome;
The present invention relates to a method for expressing a useful protein for cultivating the transformed plant source.

Furthermore, the present invention relates to the use of the above-mentioned plastid sigma factor-binding protein for promoting the expression of a useful protein in plant cells, microorganisms or animal cells, particularly in plants.

The present invention further relates to proteins that can be activated by binding a plastid genome promoter to a plastid RNA polymerase.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION The plastid sigma factor-binding protein of the present invention includes a polypeptide represented by SEQ ID NO: 2, and the sequence may be one or several as long as it retains plastid sigma factor binding activity It includes polypeptides in which a plurality of amino acids have been substituted, deleted or added.

[0014] The degree of "deletion, substitution or addition of amino acids" and their positions are determined by the modified polypeptide.
Plastid sigma factor binding activity, and thus D1 or D2
The promoter is not particularly limited as long as it has an activity of increasing the affinity of plastid RNA polymerase for the promoter of D2, preferably the promoter of D2. In the present invention, the term “plastid sigma factor binding activity” refers to an activity in which the protein or polypeptide represented by SEQ ID NO: 1 binds to a plastid sigma factor, thereby activating the D1 or D2 promoter, The expression efficiency of the useful protein incorporated later can be increased.

Specific means for substitution, addition, and deletion include:
When the method is carried out via DNA encoding the peptide, for example, cytospecific mutagenesis [Methods in Enzymology, 154, 350, 367-382 (198
7); ibid., 100, 468 (1983); Nucleic Acids Res., 12, 94
41 (1984); Laboratory of Seismic Chemistry Experiment 1, Genetic Research Method II,
Genetic engineering techniques such as the Japanese Biochemical Society, p105 (1986)], and chemical synthesis means such as the phosphate triester method and the phosphate amidite method [J. Am. Chem. Soc., 89, 4801 (1967);
Id. 91, 3350 (1969); Science, 150, 178 (1968); Tetr
ahedron Lett., 22, 1859 (1981); 24, 245 (198
3)] and methods of combining them. More specifically, the synthesis of DNA can be performed by a chemical synthesis using a phosphoramidite method or a triester method, or can be performed on a commercially available automatic oligonucleotide synthesizer. The double-stranded fragment is synthesized chemically to produce a single-stranded fragment, either by synthesizing the complementary strand and annealing the strands together under appropriate conditions, or adding the complementary strand using a DNA polymerase with the appropriate primer sequences. It can also be obtained from things. Furthermore, the polypeptide of the present invention can also be synthesized by solid phase synthesis using a peptide synthesizer, and substitution, addition, and deletion can be performed by changing the type of protected amino acid when using a peptide synthesizer. It can be done easily. Special amino acids such as D-amino acids and sarcosine (N-methylglycine) can also be introduced.

The useful protein incorporated after the D1 or D2 promoter (especially the D2 promoter) or an analog thereof is not particularly limited, and may be a useful protein for the plant itself (eg, a protein that promotes plant growth, a pest or a pathogen). Proteins that increase resistance (including antibacterial proteins, antiviral proteins, pest repellent proteins, etc.), proteins that improve the nutritional status of plants, etc.), and proteins that are useful to humans and animals that consume plants (measles, etc.) And a group of enzymes involved in the synthesis of a useful substance for obtaining a plant rich in a useful substance such as an essential amino acid (for example, lysine). Further, the use of the gene of the present invention to express a specific gene (group) in a plastid and modify photosynthetic ability, carbonic acid fixing ability, nitrogen assimilation ability, stress tolerance, etc. is also within the scope of the present invention.

The gene of the present invention is specified as a gene having an open reading frame encoding 151 amino acids represented by SEQ ID NO: 1.

The polypeptide of the present invention has a property of binding to a plastid sigma factor, for example, T3K9.5 (ge
nebank accession no. AC004261; SEQ ID NO: 4) was confirmed to bind to a plastid sigma factor.

Plants to which the protein or gene of the present invention is applied include all plants such as mosses, ferns, gymnosperms and angiosperms, and cyanobacteria. For example, various vegetables, fruits, flowers, trees, grasses, cereals and the like are exemplified.

In the present specification, the transformed plant source includes seeds, seedlings, calli, cultured cells, and the like.
In addition to plant sources, unicellular organisms having photosynthetic ability, such as chlorella, and prokaryotes, such as cyanobacteria, are also included.

In the present specification, abbreviations for amino acids, peptides, base sequences, nucleic acids, and the like are used for IUPAC, IUPAC
The rules of UB, “Guidelines for preparation of specifications including base sequence or amino acid sequence” (Japan Patent Office), and commonly used symbols in the art shall be followed.

In the present invention, a gene (DNA) is
It is intended to include not only double-stranded DNA but also single-stranded DNAs such as a sense strand and an antisense strand constituting the double-stranded DNA, and the length is not limited at all.

[0023] In the gene of the present invention, for example SEQ ID NO: gene can be exemplified having a nucleotide sequence encoding a protein comprising the amino acid sequence shown in 1,
Without being particularly limited to this, homologues of the gene are also included.

The term "homologous gene" as used herein means having sequence homology to the gene of the present invention (or a gene product thereof), and having the above-mentioned structural characteristics and similarity of its biological function as described above. It refers to a series of related genes recognized as one gene family, and naturally includes alleles (alleles) of the gene.

Specifically, a protein having a certain modification in the specific amino acid sequence represented by SEQ ID NO: 1 and having the same sigma factor binding activity as that of the protein having the specific amino acid sequence, and And a gene encoding a protein having the property of activating the promoter of D2 and increasing the expression of a gene arranged downstream of the promoter.

The homology in the above amino acid sequence is as follows:
Genome Net: Measurement using the FASTA program using Genome Net (Clustal, V., Methods Mol. Bio
l., 25, 307-318 (1994)), the total amino acid sequence can usually be about 45% or more, preferably about 50% or more.

The modification (mutation) of the amino acid sequence is
Although it may occur in nature due to, for example, mutation or post-translational modification, it can also be artificially modified based on a naturally-occurring gene. The present invention encompasses all modified genes having the above-mentioned properties, irrespective of the cause and means of such modification / mutation.

As a specific embodiment of the gene of the present invention, a gene having the base sequence shown in SEQ ID NO: 3 can be exemplified. The coding region in this nucleotide sequence corresponds to SEQ ID NO:
1 shows an example of one combination of codons indicating each amino acid residue of the amino acid sequence shown in FIG. The gene of the present invention
Not only the gene having such a specific base sequence but also a base sequence selected by combining arbitrary codons for each amino acid residue is also possible. Codon selection can be performed according to a conventional method, for example, considering the codon usage frequency of the host to be used (Nucleic Acids Res.,
9, 43 (1981)].

Further, as described above, the DNA molecule of the present invention
Also included are those consisting of a base sequence having a certain homology with the base sequence shown in SEQ ID NO: 3.

The above homology is at least 70% identity, preferably at least 90%, to a polynucleotide encoding a polypeptide containing the amino acid sequence shown in SEQ ID NO: 2 or the base sequence shown in SEQ ID NO: 3. And more preferably at least 95%, even most preferably at least 97%, and its complementary strand polynucleotide.

As such a gene, for example, 0.1%
50 ° C. or 0.1% SD in 0.2 × SSC containing SDS
14-469 in the base sequence shown in SEQ ID NO: 1 under stringent conditions at 60 ° C. in 1 × SSC containing S
And a gene having a nucleotide sequence that hybridizes with a DNA consisting of the following nucleotide sequence.

The protein of SEQ ID NO: 2 of the present invention includes not only the D1 and D2 promoters of the chloroplast genome but also the rR
Activating other promoters such as NA has been suggested by the inventors' studies (data not shown),
The protein of the present invention can activate various promoters of chloroplast genome by binding to plastid RNA polymerase.

Further, the chloroplast translocation signal of the protein of the present invention is replaced with a chloroplast translocation signal such as RbcS, or a chloroplast translocation signal such as RbcS is further added to the N-terminal side of the protein of the present invention. Can also. In addition, a polypeptide having a function other than the function of the present protein such as DNA binding activity can be added to and inserted into the protein of the present invention.

According to the present invention, it has been found for the first time that the protein of the present invention activates chloroplast promoters such as the promoter of D1 gene and the promoter of D2 gene. Are all within the scope of the present invention. When the protein of the present invention is used for activating a chloroplast promoter such as the promoter of D1 gene and the promoter of D2 gene, it is preferable to use the protein or gene of the present invention under the following conditions.

The gene of the present invention can be conveniently expressed by incorporating it into the nuclear genome or plastid genome (see below), but it is self-replicatable having a replication origin that functions in plastids (or in prokaryotes such as cyanobacteria). The expression cassette of the gene of the present invention may be incorporated into a plasmid and introduced into a plastid. It is also conceivable that the gene of the present invention is expressed by integrating it into the genome of plant-infectious virus (eg, CMV, TMV, etc.) and its analogs and infecting the virus. The promoter for expression of the gene of the present invention may be a constant promoter (CaMV35S promoter or the like on the nuclear side),
When it is necessary to identify the expression of the gene of the present invention at a certain time in a certain tissue, a promoter which can be induced by hormones or light or a promoter which shows tissue specificity may be used. In the latter case, expression of the native gene of the present invention can be eliminated by using a gene in which the native gene of the present invention is deleted. Such a deletion is obtained, for example, by searching T-DNA and transposon inserted in the gene of the present invention in higher plants, and a technology for deleting a specific gene by a conventional method has been established in cyanobacteria and the like. ing. In addition to selecting a promoter for expression of the gene of the present invention itself, naturally, a poly-A signal as a transcription terminator or post-transcriptional control,
A translation activating sequence, an mRNA stabilizing sequence and the like can be used. Furthermore, it is conceivable to introduce an intron sequence into the gene of the present invention and apply control during the process of producing mature mRNA. When the gene of the present invention is expressed in plastids, the adenine and thymine contents of the gene of the present invention can be changed to correspond to those of the plastid, and it is desirable to omit the region encoding the chloroplast translocation signal. In order to control the expression amount, timing and tissue, an appropriate promoter, translation control sequence, mRNA stability control sequence and the like can be selected and used. It is also conceivable to apply expression control by editing. When the gene of the present invention is used to activate a promoter on the chloroplast genome, it may be desirable to regulate the expression of the target sigma factor in the same manner as in the method of expressing the gene of the present invention. At this time, the introduction position of the sigma factor expression cassette is not limited, and it can be inserted into the nuclear genome or chloroplast genome, or can be introduced into the chloroplast by incorporating it into a plasmid that replicates itself in the chloroplast. It can also be used by integrating it into the genome of plant-infectious virus and its analogs. When it is desired to suppress the expression of the target sigma factor for a specific period, tissue, or condition, a deletion of the sigma factor gene is obtained in the same manner as the method for obtaining a deletion of the gene of the present invention. Then, the sigma factor may be expressed by the above-described method or the like. Also,
A similar technique can be applied to the case where the expression of a sigma factor gene that is not a target is eliminated. Alternatively, non-targeted sigma factor gene antisense RNA or double-stranded RNA
It can be expected that expression of (loop-forming RNA) can also suppress its expression.

The gene of the present invention can be prepared based on the sequence information of a specific example of the gene of the present invention disclosed by the present invention.
It can be easily produced and obtained by general genetic engineering techniques [Molecular Cloning 2d Ed, Cold Spring H
arbor Lab. Press (1989); see Seismic Chemistry Laboratory Course "Gene Research Methods I, II, III", edited by The Biochemical Society of Japan (1986), etc.)

Specifically, a cDNA library is prepared from an appropriate plant source in which the gene of the present invention is expressed in accordance with a conventional method, and the library is prepared from the library by using an appropriate probe or antibody specific to the gene of the present invention. Acad. Sci., US Proc. Natl. Acad.
A., 78, 6613 (1981); Science, 222, 778 (1983)].

In the above, the origin of the cDNA is as follows:
Examples include various cells and tissues expressing the gene of the present invention, and cultured cells derived therefrom. Separation of total RNA therefrom, separation and purification of mRNA, acquisition of cDNA, and cloning thereof can all be carried out according to conventional methods.

The method for screening the gene of the present invention from a cDNA library is not particularly limited, either, and any conventional method can be used.

As the probe used herein, DNA or the like chemically synthesized based on the information on the nucleotide sequence of the gene of the present invention can be generally used, but the gene of the present invention or a fragment thereof which has already been obtained can also be used. Good use. In addition, sense primers and antisense primers set based on the nucleotide sequence information of the gene of the present invention can be used as screening probes.

The nucleotide sequence used as the probe is a partial nucleotide sequence corresponding to SEQ ID NO: 2, and comprises at least 15 continuous bases, preferably 20 continuous bases, more preferably 30 continuous bases. Bases, most preferably those having 50 contiguous bases, are also included, or the positive clones themselves having the above sequences can also be used as probes.

When obtaining the gene of the present invention, PCR
DNA / RN by the method [Science, 230, 1350 (1985)]
The A amplification method can be suitably used. Particularly, when it is difficult to obtain a full-length cDNA from the library, RACE
Method [Rapid amplification of cDNA ends; Experimental Medicine, 12
(6), 35 (1994)], especially the 5′-RACE method [MA Frohma
n, et al., Proc. Natl. Acad. Sci., USA., 8, 8998
(1988)].

The primers used when employing the PCR method can be appropriately set based on the sequence information of the gene of the present invention revealed by the present invention, and can be synthesized according to a conventional method. The isolation and purification of the amplified DNA / RNA fragment can be carried out according to a conventional method as described above, for example, by gel electrophoresis.

The gene of the present invention or various DNA fragments obtained above can be obtained by a conventional method, for example, the dideoxy method [Proc.
Natl. Acad. Sci., USA., 74, 5463 (1977)) and the Maxam-Gilbert method (Methods in Enzymology, 65, 499).
(1980)], or simply using a commercially available sequencing kit or the like.

According to the gene of the present invention thus obtained, the presence or absence of the expression of the gene of the present invention in an individual or various tissues can be specifically determined by using, for example, a part or the entire nucleotide sequence of the gene. Can be detected.

Such detection can be carried out according to a conventional method. For example, RT-PCR [Reverse transcribed-Polyme
rase chain reaction; ES Kawasaki, et al., Amplif
ication of RNA.In PCR Protocol, A Guide to method
s and applications, AcademicPress, Inc., SanDiego, 2
RNA amplification and Northern blotting analysis [Molecular Cloning, Cold Spring Harbor La.
b. (1989)], in situ RT-PCR [Nucl.
s., 21, 3159-3166 (1993)] and cell-level measurements using in situ hybridization, NAS
BA method (Nucleic acid sequence-based amplification,
Nature, 350, 91-92 (1991)] and various other methods. Preferably, a detection method by RT-PCR can be mentioned. The primer used in the case of employing the PCR method is not particularly limited as long as it is specific to the gene of the present invention and can specifically amplify only the gene of the present invention. It can be set appropriately. Usually 1 as primer
Those having a partial sequence of the gene of the present invention having a length of about 0 to 35 nucleotides, preferably about 15 to 30 nucleotides can be mentioned. The gene of the present invention or a homologous gene or a modified gene of the gene obtained above can be used by integrating it into a nuclear genome of a plant or a cyanobacterium. Alternatively, in the case of a higher plant (having a chloroplast genome) containing ferns and mosses, it can be used by incorporating it into the chloroplast genome. It is also conceivable to incorporate the expression cassette of the present gene into a plasmid having an origin of replication that functions in chloroplasts, and to introduce it into chloroplasts. It can also be incorporated into a plant-infectious viral genome and used. When it is desired to suppress the expression of the target sigma factor for a specific period, tissue, or condition, a deletion of the sigma factor gene is obtained in the same manner as the method for obtaining a deletion of the gene of the present invention. Then, the sigma factor may be expressed by the above-described method or the like. A similar technique can be applied to the case where the expression of a non-target sigma factor gene is eliminated. Alternatively, it can be expected that the expression of antisense RNA or double-stranded RNA (RNA forming a loop) of a sigma factor gene that is not a target can also be suppressed. On the other hand, in the chloroplast genome (in the case of cyanobacteria, etc., there is no chloroplast genome, so it will be incorporated into the nuclear genome), which encodes any useful protein or polypeptide downstream of the D2 or D1 protein gene promoter. An expression cassette connecting the genes can be incorporated. Alternatively, the expression cassette can be integrated into a plasmid having an origin of replication that functions in the chloroplast and introduced into the chloroplast. It can also be incorporated into a plant-infectious viral genome and used. When it is desired to suppress the expression of the target sigma factor for a specific period, tissue, or condition, a deletion of the sigma factor gene is obtained in the same manner as the method for obtaining a deletion of the gene of the present invention. Then, the sigma factor may be expressed by the above-described method or the like. A similar technique can be applied to the case where the expression of a non-target sigma factor gene is eliminated. Alternatively, expression of antisense RNA or double-stranded RNA (RNA forming a loop) of a sigma factor gene that is not a target can be expected to suppress the expression thereof.
Preferably, the promoter of the gene encoding the native D2 or D1 protein is replaced with another promoter (promoter such as rrn16, rbcL) in order to prevent the expression of the native D2 or D1 protein from being increased by the sigma factor binding protein. . According to the transformed plant as described above, highly efficient expression of any useful gene in chloroplasts can be realized. Integration into the nuclear genome can be performed by a standard method (Plant molecular biology ap
ractical approach, edited by CH Shaw, IRL press, 1
31-220 (1988); see Cell Engineering Separate Volume, Plant Cell Engineering Series 4, Experimental Protocols for Model Plants, edited by Rice and Arabidopsis, edited by Isao Shimamoto, Kiyotaka Okada, Supervisor, Shujunsha, 78-140, etc.) For example, transformation with Agrobacteria [see the above literature and Plant Journal, 6, 27
1-282 (1994); Plant Journal 5, 551-558 (1994); Meth
ods in Plant Molecular Biology, Mary A. Schuler &
Raymond E. Zielinski, Academic Press 145-156 (198
9) etc.] and direct DNA to cells using electroporation, prokaryotic / eukaryotic conjugation transfer, DEAD dextran method, calcium phosphate method, polyethylene glycol method, particle gun (gene gun) method, etc. Introduction method [References and Plant Physiology, 93, 857-863 (199
0), Theor.Appl.Genet., 53, 57-63 (1978), Virolog
y, 52, 456 (1973), Proc. Natl. Acad. Sci. USA, 86,
6077 (1989), Jpn.J. Genet., 65, 323 (1990), Curr.
Genet., 21, 101 (1992), etc.]. The expression cassette to be incorporated is a known plasmid (see the above-mentioned literature, for example, pBI121, Clontech, pMAT (Proc.
Natl. Acad. Sci. USA, 88, 834-83 (1991), etc.)) and the like, and can be produced by a conventional method. Actually, since the sequences described below have already been incorporated into these plasmids except for the gene, it is most convenient to incorporate the sequence encoding the sigma factor-binding protein into these plasmids. Is obtained. The plasmid already contains the genes required for integration into the nuclear genome (such as the ver gene).
The portion to be integrated into the nuclear genome is sandwiched between sequences for that purpose (T-DNA Rightborder (RB), Left border (LB), etc.). The expression cassette of the gene will be inserted into this region. In this cassette, the gene is tethered downstream of a promoter (eg, a CaMV35S promoter, a hormone-inducible promoter, or in the case of prokaryotes such as cyanobacteria, a prokaryotic promoter), a transcription terminator, a poly-A signal (T- NOS, etc.) are linked downstream of the gene (poly-A signal is only for eukaryotes). The integrated region (the region between RB and LB) can contain an expression cassette for a drug resistance gene (for example, a kanamycin resistance gene or a hygromycin resistance gene) for selecting a transformant. Although this drug resistance gene is often integrated into the above-mentioned plasmid, its removal or replacement with another resistance gene can be performed by a conventional method. When a drug resistance gene for selecting a transformant has been incorporated, it is simply performed by selecting a plant that grows in the presence of an appropriate drug. Conventional methods such as Southern hybridization, Western blotting, and PCR can be used for confirming the transformants and selecting the transformants in which the drug resistance gene is not inserted. The selected plants are often heterozygous, but homologous individuals can be obtained by self-pollination or the like. If you integrate the sigma factor binding protein gene into the plastid (chloroplast) genome instead of the nucleus,
The introduction into the plastid genome can be performed by the direct DNA introduction method (polyethylene glycol method or particle gun method (Pro
c. Natl. Acad. Sci. USA, 90, 913-917 (1993)); see Examples) (see Examples). In this case, a promoter (rrn1) that functions in the chloroplast as a promoter for expressing the gene in the expression cassette
6, using a translation initiation sequence that functions in chloroplasts such as psbA, rbcL, etc., and an SD sequence, preferably a terminator that functions in chloroplasts (chloroplast gene psbA,
terminator such as rps16 or a prokaryotic type terminator). In addition, as a drug resistance gene,
It can also contain a spectinomycin resistance gene and the like (see Examples). In order to integrate this expression cassette into the chloroplast genome by homologous recombination, a sequence at an appropriate position in the chloroplast genome (position to be integrated) is added to both sides of the present expression cassette (see Examples). Transformants containing the expression cassette were selected by culturing in the presence of the drug (Pro
c. Natl. Acad. Sci. USA, 87, 8526-8530 (1990)), Southern hybridization, Northern hybridization, Western blotting, PCR method and the like, and confirm the transformant. In many cases, the resulting transformants are heterozygous plants in which some of the plastid genomes have been transformed.If necessary, selection with a drug was repeated, and all plastid genomes were transformed. Obtain homozygous individuals. When the sigma factor-binding protein gene is introduced into a chloroplast as a plasmid that replicates autonomously in plastids, the above-described expression cassette is incorporated into the plasmid, for example, a prokaryotic / eukaryotic conjugation transfer method or a particle gun method. The above-described direct DNA transfer method is used. In this case, it is desirable that the present plasmid contains a drug resistance gene for selecting a transformant. Incorporation of any useful protein gene into the chloroplast genome (in the case of cyanobacteria, etc. into the nuclear genome by the above-mentioned method), the chloroplast promoter such as D2 or D1 promoter (or a similar sequence thereof) and
It can be carried out by connecting a gene encoding any useful protein downstream of a translation initiation sequence such as an SD sequence, and preferably connecting a transcription terminator downstream thereof. More preferably, the mRNA stabilizing sequence and the translation activating sequence (5′UTR, 3′UTR, etc. of the chloroplast gene rbcL, etc.) are functionally linked to the untranslated region or translated region of the desired gene.
In addition, it is desirable that the codon of the gene be modified in accordance with the frequency of use in chloroplasts for the purpose of mass expression, and the adenine / thymine content is changed to correspond to that of the host (chloroplast, cyanobacterium). You can also. The plasmid containing the above sequence can be prepared by a conventional method. This plasmid is integrated into the chloroplast genome by the above-mentioned method to obtain a transformant. When a sigma factor-binding protein is incorporated into the chloroplast genome, the expression cassette can be connected to the expression cassette, and a transformant can be prepared by a single operation. Also, the expression cassette of the desired gene is incorporated into a plasmid that autonomously replicates in chloroplasts,
It can also be introduced into chloroplasts. In addition, an expression cassette for a sigma factor-binding protein can be simultaneously incorporated into the present plasmid. When replacing the promoter of the native D2 or D1 protein gene in the chloroplast genome, a chloroplast genome fragment obtained by replacing the promoter of the gene encoding the native D2 or D1 protein with another promoter is used. Prepared and introduced by homologous recombination into the genomic location where these promoters are present. in this case,
It is also possible to incorporate a sigma factor-binding protein expression cassette (when incorporated into a chloroplast) and / or a useful protein gene expression cassette into the chloroplast genome fragment, and obtain a transformant by a single operation. When replacing the above-mentioned native promoter in the cyanobacterial genome, a conventional method of genetic manipulation,
It can be performed by homologous recombination. The transformant obtained as described above can strongly or inducibly express the sigma factor-binding protein, and as a result, transcription of the useful protein gene linked to the promoter of the D1 or D2 protein gene, and furthermore, its expression Is expected to increase dramatically.

According to the gene of the present invention, the gene product or a protein containing the gene product can be easily and stably produced in a large amount by using ordinary genetic engineering techniques.

The protein of the present invention can be prepared by a conventional gene recombination technique (eg, Science, 224, 1431 (1984); Biochem. B) based on the sequence information of the gene provided by the present invention.
iophys. Res. Comm., 130, 692 (1985); Proc. Natl. A
cad. Sci., USA., 80, 5990 (1983) and the like].

More specifically, the production of the protein is carried out by preparing a recombinant DNA (expression vector) capable of expressing a gene encoding the desired protein in a host cell, and introducing this into a host cell for transformation. Then, the transformant is cultured, and then recovered from the resulting culture.

As the host cell, any of prokaryote and eukaryote can be used. For example, as a prokaryote host, Escherichia coli, especially Escherichia
Escherichia coli K12 strain can be exemplified. Eukaryotic host cells also include vertebrates,
Cells such as yeast and plants are included.

When a prokaryotic cell is used as a host, a promoter and an SD base sequence, as well as a promoter and an SD base sequence, are arranged upstream of the vector so that the gene of the present invention can be expressed in the vector using a vector that can be replicated in the host cell. An expression plasmid having an initiation codon (eg, ATG) required for initiation of protein synthesis can be suitably used. As the vector, generally, a plasmid derived from Escherichia coli, for example, pBR322, pBR3
25, pUC12, pUC13 and the like are often used.

Specific examples of expression vectors for expression in the cytoplasm using a plant as a host include various vectors having the CaMV35S promoter (for example, pBI121 vector (Toyobo
P35S-CAT (Plant Journal, 5, 421-427 (1994)) p
CaMV35S-sGFP (S65T) -NOS3 '(Proc. Natl. Acad. Sci. U
SA, 94, 14948-14953 (1997)) and hormone-inducible promoters.

The promoter is not particularly limited. When Escherichia is used as a host, for example, tryptophan (trp) promoter, lpp promoter, lac promoter, r
An ecA promoter, a PL / PR promoter and the like can be preferably used.

When the host is a plant, the CaMV35S promoter is generally used. When expressed in plastids, as shown in the present invention, D1 and D2 protein promoters,
In particular, the promoter of the D2 protein can be suitably used. Also, promoters of chloroplast genes such as psbA and rbcL, and prokaryotic promoters can be used.

A method for introducing a desired recombinant DNA (expression vector) into a host cell and a transformation method using the same include:
There is no particular limitation, and various general methods can be employed.

The obtained transformant can be cultured according to a conventional method, and the target protein of the present invention encoded by a gene designed as desired by the culture can be cultured in the cell, extracellularly or on the cell membrane of the transformant. Expression, production (accumulation, secretion)
Is done.

As the medium to be used for the culture, various media commonly used depending on the host cell used can be appropriately selected and used, and the culture can be carried out under conditions suitable for the growth of the host cell.

The thus obtained recombinant protein of the present invention can be subjected to various separation operations utilizing its physical properties, chemical properties, etc. [Biochemical Data Book II,
1175-1259, 1st edition, 1st printing, June 23, 1980, published by Tokyo Chemical Co., Ltd .; Biochemistry, 25 (25), 8274 (198
6); Eur. J. Biochem., 163, 313 (1987)].

Specific examples of the method include ordinary reconstitution treatment, treatment with a protein precipitant (salting out method), centrifugation,
Osmotic shock method, sonication, ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, high-performance liquid chromatography (HPL
Various types of liquid chromatography such as C), dialysis, and combinations thereof can be exemplified. Particularly preferred methods include affinity chromatography using a column to which a specific antibody against the protein of the present invention is bound. it can.

In addition, in the amino acid sequence encoded by the gene, when a part of the amino acid residues or the amino acid sequence is modified by substitution, deletion, addition, etc., the amino acid sequence may be modified by various methods described above such as cytospecific mutagenesis. It can be done by a method.

The protein of the present invention can be produced by a general chemical synthesis method according to the amino acid sequence shown in SEQ ID NO: 1. The method includes a conventional liquid phase method and a solid phase method for peptide synthesis.

The peptide synthesis method is described in more detail
Based on the amino acid sequence information, a so-called stepwise elongation method in which each amino acid is sequentially linked one by one to extend the chain, a fragment consisting of several amino acids is synthesized in advance, and then each fragment is subjected to a coupling reaction. And the fragment condensation method, and the synthesis of the protein of the present invention may be carried out by any of them.

The protein of the present invention thus obtained is appropriately purified according to the above-mentioned various methods, for example, a method widely used in the field of peptide chemistry such as ion exchange resin, partition chromatography, gel chromatography, and countercurrent partition method. It can be carried out.

[0064]

According to the present invention, proteins and / or genes discovered for the first time in the present invention can be used to express any useful protein in plants with high efficiency.

[0065]

The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the present invention is limited to these Examples. (1) Expression of GFP in chloroplast-chloroplast transformation technique The present inventors have studied psbA promoter and GFP (green fluorescent pr
otein: green fluorescent protein) to obtain a stable transformant carrying the fusion gene in the chloroplast genome.
GFP was successfully expressed (Plant Cell Physiology, 4, 367-3
71 (2000)). Tobacco psbA promoter, SD sequence, gfp
It was connected upstream of a GFP cassette consisting of the gene, Trps16 (terminator). Here, the gfp gene has a mutation introduced into the chromophore region (Phe64Ser65 is replaced with Leu64Thr65). This is called pRV112A (Nucl. Acids. Res.
22, 3819-3824), which was incorporated into a chloroplast transformation vector pKH5 plasmid. The pKH5 plasmid has an aadA cassette containing the spectinomycin resistance gene aadA. The aadA cassette is [promoter of 16S rDNA :: aadA ::
psbA terminator], pCT08 (Proc. N
Atl. Acad. Sci. USA, 95, 9705-9709 (1998)). Transformation of tobacco chloroplasts was performed by injecting the plasmid prepared as described above into a sterile leaf with a particle gun (Biorad, PDS1000He Bi
olistic gun; Proc. Natl. Acad. Sci. USA, 90, 913-9
17 (1993)). Subsequently, transformation shoots were selected on a medium containing spectinomycin (Proc. Natl. Acad. Sc).
i. USA, 87, 8526-8530 (1990)). The transformant was confirmed by Southern hybridization, and it was confirmed that the transformant had the correct inserted sequence at the target position. In addition, homozygotes in which all chloroplast genomes were transformed could be obtained. The expression of gfp mRNA was confirmed by Northern hybridization. Expression of GFP was determined by confocal laser microscopy (BioRad, MRC600 Z or micro-R
adiance). GFP was accumulated only in the chloroplast, and a tubular structure was observed.

The above results demonstrate that a foreign gene can be incorporated and expressed in chloroplasts. (2) Identification and functional analysis of sigma factor binding protein The present inventors targeted a sigma factor binding protein that has not been found in eukaryotes.

[0067] plastid sigma factor that is reported to date, four conserved sequences are those homologous to class I and II of bacteria, including E. coli sigma ⁷⁰ (region from the region 1 4)
Having. In particular, in the promoters of the genes psbA and psbD encoding the D1 and D2 proteins, the -35 element is not required or not present for light-responsive transcription,
The present inventors have focused on a region 4 which is known to recognize -35 elements in E. coli sigma ^70.

The present inventors have proposed the yeast two-hybrid method (Clonte
ch, Yeast Protocols Handbook (PT3024-1), Matchmake
r See the Gal4 two-hybrid user manual (PT3061-1), etc.) to determine the plastid sigma factor Arabidopsis thaliana SigA
(FEBS Letters, 413, 309-313 (1997), Proc. Natl. A
cad. Sci. USA, 94, 14948-14953 (1997), etc.). Two positive clones were obtained from 2 × 10 ⁶ clones, which encoded the same protein.

The Arabidopsis thaliana cDNA library was screened using cDNA fragments obtained from the positive clones described above, and a full-length cDNA was identified. The largest open reading frame of the longest cDNA encoded a 151 amino acid sequence (FIG. 1 and SEQ ID NO: 2). This protein is abbreviated as “SibI”. Database search revealed that Sibl was not homologous to any protein with a known function. However, one homolog with 56.3% homology was found in Arabidopsis (T3K9.5, genebank acce).
ssion no. AC004261 and SEQ ID NO: 4).

The N-terminal region of Sibl is positively charged and rich in amino acid residues having a hydroxyl group (SEQ ID NO: 1), which is a chloroplast translocation signal, a transit peptide (trans
It peptide). On the other hand, PSORT (protein classification program, http://psort.nibb.ac.jp)
Predict a nuclear localization signal within the terminal region. To examine the transport of SibI into chloroplasts, two plasmids were
GF fused to the N-terminal 40 amino acids (TP40) or 92 amino acids (TP92) of Sibl under the control of the 35S-promoter
P (K. Isono et al., Proc. Natl. Acad. Sci. USA 94, 149
48 (1997)) (each GFP fusion protein will be referred to as TP40, TP92). GFP only or RbcS transit peptide (W.-L. Chiu et al., C
Plasmids expressing GFP fused with urr Biol., 6, 325 (1996)) were used as chloroplast transport negative and positive controls, respectively (referred to as GFP and RBCS, respectively). Protoplasts prepared from Arabidopsis rosette leaves were subjected to the polyethylene glycol method (S. Abel, A. Theologis, Plan
t J. 5, 421 (1994)).
The position of GFP fluorescence was examined (FIG. 2).

In the negative control expressing GFP, no GFP signal was observed in the chloroplast, indicating that GFP was not transported to the chloroplast without the transit peptide. In the case of TP92 and RBCS (positive control), the transformed cells showed GFP fluorescence from chloroplasts,
Chloroplast transport was not detected in cells expressing TP40. These results indicate that the N-terminal 92 amino acids function as a transit peptide, but the N-terminal 40 amino acids do not function as a transit peptide.
However, the subcellular distribution of endogenous SibI should be determined. This is because TP40 and TP92 have a higher tendency to localize at or around the nucleus than control GFP (data not shown).

In order to elucidate the function of SibI in plastid gene transcription, the present inventors have established a chloroplast transcription run-on assay system using a transformed protoplast. Protoplasts prepared from 7-week-old rosette leaves were transformed with a plasmid constructed to express the entire coding region of Sibl under the control of the CaMV35S promoter. A plasmid expressing TP92 instead of full-length Sibl (Figure 2) was used for control experiments. The transformed protoplasts were CBS (0.3 M sorbitol, 50 mM He
pes-NaOH, pH 7.6, 2 mM EDTA) to give a chlorophyll concentration of 2.5 mg / ml. This protoplast suspension was mixed with a run-on mixture (37.5 mM Hepes-NaOH, pH 7.9, 12.5 mM Mg
Cl ₂ , 50 mM KCl, 25 μg / ml heparin, 625 μM each ATP, CT
P and GTP, 62.5 μM UTP, 200 μCi [α- ³² P] UTP, 240 U R
Nase inhibitors) and the labeled transcripts were analyzed by hybridizing to a dot-blotted membrane with DNA of a representative chloroplast gene. FIG. 3 compares the resulting transfer patterns with protoplasts treated in three different ways. In untransformed protoplasts, rrn16, rbcL, and ps in the chloroplast gene
The relatively high transcription of bA has been reported in previous studies (J. Biol. Chem. 26
7, 21404-21411 (1992), Plant Physiol. 109, 105-112
(1995)). Surprisingly, dramatic activation of psbD transcription and strong enhancement of psbA transcription
Found in bI-expressing protoplasts, but not in TP92-expressing protoplasts. Transcription of other plastid genes was hardly affected by Sibl. These results indicate that transcriptional enhancement is induced by Sibl and is specific for the psbD and psbA promoters.

To test the specific binding of SibI to SigA, SibI and other sigma factors in Arabidopsis (SigB,
Examination of the interaction between SigD and SigE) by the yeast two-hybrid method showed that no interaction was observed except for SigA (data not shown). In order to confirm the direct interaction between SibI and SigA region 4, a GST pull-down assay was performed as shown in FIG.

MBP-SibI (SibI and maltose binding protein
n (MBP) fusion protein) is GST-SigAR4 (SigA region 4
Interacts only with GST (Glutathione-S-transferase (GST)) (lane 5), and does not react with GST (lane 6) or GST-SigBR4 (lane 4). The results shown in FIG. 4 indicate that the interaction is direct and that the interacting moiety is on a portion of SibI and SigA region 4.

Consistent with the results of the yeast two-hybrid assay, the results of the pull-down assay clearly show that SibI and SigA region 4 interact directly and specifically.

By performing the same experiment using T3K9.5 in place of SibI, T3K9.5 was found to have SigA in the same manner as SibI.
(Data not shown).

Finally, the present inventor has reported that the psbD promoter is Si
It emphasizes that in combination with bI, it offers a very high potential to be a very strong promoter in the construction of a chloroplast factory of the desired useful protein. (3) Sigma factor binding protein gene (SibI, T3K9.5
The present inventors have prepared a transformed plant that expresses the sense (and antisense) strands of the Sibl and T3K9.5 genes.
This experiment was performed for the purpose of trying to express large amounts of SibI or T3K9.5 in a plant body (and to suppress the expression). These genes were incorporated into a pBI121 plasmid (binary vector, Clontech) so as to express the sense strand (and antisense strand) under the CaMV35S promoter, and to place the nos3 'region downstream, and to transform Agrobacteria. Converted. Kanamycin was used for selection of Agrobacterium transformants. Arabidopsis was infected with the transformed Agrobacteria by vacuum infiltration (Cell Engineering Separate Volume, Plant Cell Engineering Series 4, Experimental Protocol for Model Plants, Rice and Arabidopsis thaliana, edited by Isamu Shimamoto, supervised by Kiyotaka Okada, Shujunsha 109-113) . Recovering seeds from infected plants,
The cells were seeded on a selection medium containing kanamycin to obtain a transformant. It was also possible to collect those next-generation seeds. The above results suggest that strains overexpressing and suppressing SibI or T3K9.5 have no abnormality in reproductive function.

[0078]

[Sequence List] SEQUENCE LISTING <110> KANSAI TLO Co., ltd. <120> Plastid Sigma Factor Binding Protein <130> 10F00JP <160> 4 <170> PatentIn Ver. 2.0 <210> 1 <211> 754 <212> DNA <213> Arabidopsis thalina <400> 1 acaggaaccg aac atg gag tca tca tcg tcg act ttt ctc acc acg aca 49 Met Glu Ser Ser Ser Ser Thr Phe Leu Thr Thr Thr 1 5 10 agc tta gac aag aaa aaa cca tct ccg gtt tca aga aaa tca cca aag 97 Ser Leu Asp Lys Lys Lys Pro Ser Pro Val Ser Arg Lys Ser Pro Lys 15 20 25 caa aag aag aag acg act tcg acg aat aaa ccc atc aaa gtt cgt tac 145 Gln Lys Lys Lys Thr Thr Ser Thr Asn Lys Pro Ile Lys Val Arg Tyr 30 35 40 atc tca aac ccc atg aga gtt caa act tgt gct tct aag ttc aga gag 193 Ile Ser Asn Pro Met Arg Val Gln Thr Cys Ala Ser Lys Phe Arg Glu 45 50 55 60 ctc gtt caa gaa ctc aca ggc caa gac gcc gtc gat ctt caa ccg gag 241 Leu Val Gln Glu Leu Thr Gly Gln Asp Ala Val Asp Leu Gln Pro Glu 65 70 75 ccc atc tat tcc cct tcc tcc gac gac cac aac ctt tct cca cc gcg 289 Pro Ile Tyr Ser Pro Ser Ser Asp As p His Asn Leu Ser Pro Pro Ala 80 85 90 gag aat ctt gcg cca cgt gtt ctt cat cag gag ccg ttc ggt gag aga 337 Glu Asn Leu Ala Pro Arg Val Leu His Gln Glu Pro Phe Gly Glu Arg 95 100 105 gac agt gat tgt tat gag ccg ttg aat gcg gaa gac atg ttt tta cct 385 Asp Ser Asp Cys Tyr Glu Pro Leu Asn Ala Glu Asp Met Phe Leu Pro 110 115 120 gat cag atg tcg gca ggt ttt tct ggg ttt ttc tct aat ggg ttt tac Asp Gln Met Ser Ala Gly Phe Ser Gly Phe Phe Ser Asn Gly Phe Tyr 125 130 135 140 aat gtc aat gac ttt gga agc atc gat tct atg tgataatctt 476 Asn Val Asn Asp Phe Gly Ser Ile Asp Ser Met 145 150 151 tcagagtttt tattagggt tattaggg agatatatat cattatgata tcgatacata 536 aatttaaccc ccaaattgat acatggatat gttaattact atagggtttg ggatgcgaat 596 tagtatatga gtaagttgtt ttgtaaacag tttttttttt cttaaatttg tttgtttgtt 656 ttttcttaca tcaaaacttc ggtaatgtat gaacatattt tctatttaat caagaaacat 716 gtgataatct aaagttagcg attaataagc cttcaagt 754 <210> 2 <211> 151 <212> PRT <213> Arabidopsis thalina <400> 2 Met Glu Ser Ser Ser Ser Thr Phe Leu Thr Thr Thr Ser Leu Asp Lys 1 5 10 15 Lys Lys Pro Ser Pro Val Ser Arg Lys Ser Pro Lys Gln Lys Lys Lys 20 25 30 Thr Thr Ser Thr Asn Lys Pro Ile Lys Val Arg Tyr Ile Ser Asn Pro 35 40 45 Met Arg Val Gln Thr Cys Ala Ser Lys Phe Arg Glu Leu Val Gln Glu 50 55 60 Leu Thr Gly Gln Asp Ala Val Asp Leu Gln Pro Glu Pro Ile Tyr Ser 65 70 75 80 Pro Ser Ser Asp Asp His Asn Leu Ser Pro Pro Ala Glu Asn Leu Ala 85 90 95 Pro Arg Val Leu His Gln Glu Pro Phe Gly Glu Arg Asp Ser Asp Cys 100 105 110 Tyr Glu Pro Leu Asn Ala Glu Asp Met Phe Leu Pro Asp Gln Met Ser 115 120 125 Ala Gly Phe Ser Gly Phe Phe Ser Asn Gly Phe Tyr Asn Val Asn Asp 130 135 140 Phe Gly Ser Ile Asp Ser Met 145 150 151 <210> 3 <211> 754 <212> DNA <213> Arabidopsis thalina <400> 3 acaggaaccg aacatggagt catcatcgtc gacttttctc accacgacaa gcttagacaa 60 gaaaaaacca tctccggttt caagaaaatc accaaagcaa aagaagaaga cgacttcgac 120 gaataaaccc atcaaagttc gttacatctc aaaccccatg agagttcaaa cttgtgctcgag taagcgtcagagc taga c gccgtcgatc ttcaaccgga 240 gcccatctat tccccttcct ccgacgacca caacctttct ccaccggcgg agaatcttgc 300 gccacgtgtt cttcatcagg agccgttcgg tgagagagac agtgattgtt atgagccgtt 360 gaatgcggaa gacatgtttt tacctgatca gatgtcggca ggtttttctg ggtttttctc 420 taatgggttt tacaatgtca atgactttgg aagcatcgat tctatgtgat aatctttcag 480 agtttttatt aggggtttca tctagtagat atatatcatt atgatatcga tacataaatt 540 taacccccaa attgatacat ggatatgtta attactatag ggtttgggat gcgaattagt 600 atatgagtaa gttgttttgt aaacagtttt ttttttctta aatttgtttg tttgtttttt 660 cttacatcaa aacttcggta atgtatgaac atattttcta tttaatcaag aaacatgtga 720 taatctaaag ttagcgatta ataagccttc aagt 754 <210> 4 <211> 426 <212> DNA <213> Arabidopsis thalina T 3 K9.5 <400> 4 atg gat cag tca tca tca acg ttg ctc ata aac cag aga aag tca tca 48 Met Asp Gln Ser Ser Ser Thr Leu Leu Ile Asn Gln Arg Lys Ser Ser 1 5 10 15 tcg tct ccg act agg atc cca cct aag cag aag agg aag tca acg act 96 Ser Ser Pro Thr Arg Ile Pro Pro Lys Gln Lys Arg Lys Ser Thr Thr 20 25 30 acg cac aaa ccc at c aaa gtc cgt tac ata tca aat ccg atg aga gtc 144 Thr His Lys Pro Ile Lys Val Arg Tyr Ile Ser Asn Pro Met Arg Val 35 40 45 gag act tgt cct tct aag ttc aga gag ctt gtt caa gaa ctc acc ggt 192 Glu Thr Cys Pro Ser Lys Phe Arg Glu Leu Val Gln Glu Leu Thr Gly 50 55 60 caa gac gcc gct gat cta cca ccg tcg ccc acc act ttt acc gcc gta 240 Gln Asp Ala Ala Asp Leu Pro Pro Ser Pro Thr Thr Phe Thr Ala Val 65 70 75 80 gat ctc cac cgt ccg tgt gag tcg gag atg aac tta gag ccg ttg gat 288 Asp Leu His Arg Pro Cys Glu Ser Glu Met Asn Leu Glu Pro Leu Asp 85 90 95 gga gag gtc cgt gga gag tat tat tca ccg ttg gat gag gaa gtg ttt 336 Gly Glu Val Arg Gly Glu Tyr Tyr Ser Pro Leu Asp Glu Glu Val Phe 100 105 110 aat gct cct cag atg tcg gca ggt ctt tct ggt ttt ttc tcg tct gga 384 Asn Ala Pro Gln Seret Ala Gly Leu Ser Gly Phe Phe Ser Ser Gly 115 120 125 ttt tac aat gtt aac gct tta gga agc att ggt tct ctc tga 432 Phe Tyr Asn Val Asn Ala Leu Gly Ser Ile Gly Ser Leu * 130 135 140 141

[Brief description of the drawings]

FIG. 1 shows the amino acid sequence and base sequence of the promoter binding protein of the present invention. In FIG. 1, 16th to 32nd
The underlined amino acid sequence at the position indicates a putative nuclear localization signal. Two polyadenylation sites are indicated by *. TP40 and TP92 indicate the ends of the N-terminal region fused to GFP in FIG.

FIG. 2 is a photograph substituted for a drawing, showing chloroplast transport of SibI exhibited by the GFP fusion protein. Green channels indicate GFP signal in transformed protoplast cells. The red channel shows chlorophyll fluorescence from chloroplasts. The images were taken with a conventional fluorescent microscope,
Processed by Adobe Photoshop.

FIG. 3. ps in transformed SibI-expressing protoplasts
4 is a photograph as a substitute of a drawing, showing specific activation of bD and psbA transcription. Protoplasts and chloroplasts burst under the effect of osmotic pressure when added to a run-on mixture containing [α- ³² P] UTP. After incubation at 25 ° C. for 10 minutes, the labeled transcript was purified and some of the plastid gene was purified.
Hybridized to dot-blotted membrane with DNA
(Plastid DNA was prepared by PCR. PsbD: 925 bp (78-
1002), rbcL: 944bp (104-1047), rrn16: 923bp (363-128
5), psbA: 918 bp (106-1023), psbB: 930 bp (554-1483),
psaA: 963 bp (433-1395), rpoB: 949 bp (1795-2743). The number in parentheses is the position from the translation start site). The final washing conditions are 0.1 × SSC, 0.1% SDS at 50 ° C. for 30 minutes. The images are pictrography and BAS2000
Was obtained. lDNA acts as a negative control. "No plasmid" indicates the result of treatment using a plasmid treated in the same manner except that no transforming plasmid was added. The protoplast is Si
Transformation was performed with the plasmid constructed to express bI (method using polyethylene glycol). This plasmid contains the CaMV35S promoter, sibI, and terminator (pul
yA signal) (NOS3 ').
The plasmid used to express is the one used in FIG.

FIG. 4 is a photograph substituted for a drawing, showing a specific and direct interaction between SibI and SigA region 4. Fusion proteins of glutathione-S-transferase (GST) with SigA region 4 and SigB region 4 (GST-SigAR4 and GST-SigBR4, respectively) were expressed in E. coli and immobilized on glutathione-Sepharose4B beads. GST was immobilized on the beads as a control. SibI is expressed in Escherichia coli as a fusion protein (MBP-SibI) with maltose binding protein (MBP),
Collected in the fraction precipitated by 0% saturated ammonium sulfate. MBP-SibI in the fraction
Was one of the major proteins (lane 10). MB
P was prepared for control experiments (lane 1
1). MBP-SibI or MBP was mixed with the beads prepared above and incubated at 25 ° C. for 1 hour. Solution A
After completely washing with (20 mM Tris-HCl, pH 7.3 and 150 mM NaCl), the bound protein was removed from the glutathione solution (50 mM glutathione, 150 mM NaCl, and 100 mM Tris-HCl, pH 8.0).
And analyzed by SDS-PAGE and silver staining (lanes 4 to 9). Lanes 1 to 3 show the results of a control test performed without adding MBP-SibI or MBP.

 ────────────────────────────────────────────────── ─── Continuing on the front page (72) Inventor Yoshinori Toshima Yoshida Nihonmatsucho, Sakyo-ku, Kyoto Kyoto University Graduate School of Human and Environmental Studies F-term (reference) 2B030 AA02 AB03 AD08 CA06 CA17 CA19 CB02 CD03 CD07 CD09 4B024 AA08 BA21 BA80 CA04 DA01 DA05 EA04 FA02 GA11 GA19 HA01 HA14 4B065 AA11X AA88X AA88Y AB01 AC09 AC14 BA02 BA25 CA24 CA53 4H045 AA10 AA30 CA30 DA01 EA05 FA74

Claims (10)

[Claims] 1. A polypeptide of the following (a) or (b): (a) a polypeptide represented by SEQ ID NO: 2; (b) one or more amino acids in the polypeptide (a) are substituted or deleted. A polypeptide comprising a lost or added polypeptide and having plastid sigma factor binding activity. 2. A gene comprising any of the following polynucleotides (a) and (b): (a) a base sequence represented by SEQ ID NO: 1 or a complementary strand thereof; A polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of a base sequence. 3. The gene according to claim 2, which is a nucleotide sequence represented by SEQ ID NO: 1. 4. A gene encoding a polypeptide having the amino acid sequence represented by SEQ ID NO: 2. 5. A plastid genome having an expression cassette operably linked to a gene encoding a useful polypeptide downstream of a promoter of a D1 or D2 protein gene or an analog thereof. 6. A transformed plant source having the plastid genome according to claim 5. 7. A method for expressing a useful protein for cultivating the transformed plant source according to claim 6. 8. Use of the polypeptide according to claim 1 for promoting the expression of a useful protein in a plant. 9. A protein capable of binding to a sigma factor of an organism having photosynthetic ability. 10. The plastid genome promoter is a plastid RNA.
A protein that can be activated by binding to a polymerase.