JP4373363B2 - Long-chain DNA fragment-introduced chloroplast transformation vector - Google Patents

Description

  The present invention relates to a conversion vector capable of introducing a useful long DNA fragment of 8 kbp or more into a chloroplast genome.

In the basic research so far, a chloroplast transformation technique for introducing a foreign DNA of several kbp into a tobacco chloroplast genome has been established (for example, see Patent Document 1). In this case, a chloroplast transformation vector for carrying a target gene to a chloroplast is prepared based on a pUC vector or the like. However, conventionally, the size of foreign DNA that can be cloned, such as a pUC vector, is up to about 8 kbp. For this reason, it was impossible to insert a long-chain DNA fragment of 8 kbp or more such as fatty acid synthase into a chloroplast transformation vector.
In addition, it is known that genes for functional expression have a cluster structure due to recent progress in genome research. In many cases, the gene group having such a structure exhibits a DNA size of 8 kbp or more. For this reason, when a chloroplast transformation vector having a basic skeleton such as a pUC vector currently used is used, it is difficult to clone a gene group having a cluster structure. Moreover, the gene which has taken the cluster structure often exhibits the function as a gene group. For this reason, in a gene having a cluster structure, it is desirable to introduce a long-chain DNA fragment containing the gene group.
Regarding introduction of long-chain DNA fragments into plants, an example of successful introduction of about 80 kbp of DNA into the nuclear genome has been reported (see Non-Patent Document 1). However, in the case of gene introduction into the plant nucleus, gene transmission to nature due to pollen scattering is regarded as a problem. On the other hand, reports of DNA introduction in chloroplast transformation, which has a low possibility of gene transmission to nature due to pollen scattering, have only been reported up to a maximum of about 6 kbp (see Non-Patent Document 2). ).
JP 2000-253768 A Liu et al., Proceedings of the National Academy of Science of the United State of America (Proc. Natl Acad. Sci. USA), 1999, Vol. 96, p. 6535-6540 Lossl et al., Plant cell Rep., 2003, Vol. 21, p. 891-899

  An object of the present invention is to provide a vector capable of introducing a useful long-chain DNA fragment of 8 kb or more into a chloroplast genome.

  The present inventors inserted a DNA derived from two types of chloroplast genomic DNA into a BAC (bacterial artificial chromosome) bacterial artificial synthetic vector, and the DNA derived from the chloroplast genomic DNA was 8 kbp or more. A vector in which a useful long DNA fragment was introduced was prepared, and it was found that the vector can be introduced into the chloroplast genome of a plant leaf cell and the plant can be transformed. Based on these findings, the present inventors have further studied and completed the present invention.

That is, the present invention
[1] A chloroplast transformation vector comprising a useful long-chain DNA fragment of 8 kbp or more introduced between two types of DNA derived from chloroplast genomic DNA,
[2] In the nucleotide sequence of a region where two types of chloroplast genome DNA are linked to each other with the two structural genes on the chloroplast genome across the non-coding region, the 5 ′ end of the non-coding region 3 'side or 5' side from the 3 'end and a base sequence obtained by dividing at least about 50 bp into two structural genes from the position divided at the non-coding region, The vector according to [1], wherein each of the vectors has a length of at least about 700 bp and has at least 50 bp of a base sequence of a structural gene linked to a divided non-coding region,
[3] The two types of structural genes described above, wherein the two types of genes are selected from the combination of trnG and trnfM, rbcL and accD genes, trnI and trnA, and 3′rps12 gene and trnV. 2],
[4] A useful long-chain DNA fragment of 8 kbp or more is (a) a DNA fragment containing a fatty acid synthase gene; (b) a DNA fragment containing a structural gene of nitrogen-fixing enzyme nitrogenase; (c) Calvin involved in photosynthesis A DNA fragment ligated with a DNA encoding an enzyme constituting a cycle-limiting enzyme group; (d) a DNA fragment containing an enzyme gene involved in biosynthesis of a useful component produced in a plant; (F) a DNA fragment containing a gene encoding a herbicide-resistant protein; (g) a DNA fragment containing an environmental stress tolerance gene or a gene related to production efficiency; (h) a gene that biosynthesizes rubber A DNA fragment comprising a group; (i) a DNA fragment comprising a gene group that biosynthesizes hydrocarbons; (j) a gene group that biosynthesizes amino acids; (K) a DNA fragment containing a gene group that biosynthesizes biodegradable plastic raw materials, etc .; (l) a DNA fragment containing a cellulose synthase gene group; (m) a DNA fragment containing a polyketide compound synthase gene group (N) the vector according to any one of [1] to [3] above, which is at least one selected from DNA fragments containing a cellulolytic enzyme gene group;
[5] The vector according to any one of [1] to [3] above, wherein the introduced long DNA fragment of 8 kbp or more is a DNA fragment derived from cyanobacterial genomic DNA,
[6] A cyanobacterial genomic library prepared with the vector according to [5],
[7] A plant transformed with the vector according to any one of [1] to [5],
[8] A method for transforming a plant chloroplast characterized by introducing the vector according to any one of [1] to [5] into a plant leaf cell, and [9] The transformation method according to [8], wherein a particle gun is used,
About.

Using the long-chain DNA fragment-introduced chloroplast transformation vector constructed in the present invention, a chloroplast into which an exogenous long-chain DNA fragment has been introduced, which could not be introduced with a conventional chloroplast transformation vector. Creation of body-transformed plants has become possible.
Furthermore, the produced chloroplast transformation vector can be used for the construction of a long-chain genomic library for genome function analysis. In addition, if a vector that constructs a long-chain genome library is used, the long-chain genome can be directly introduced into the chloroplast genome.
By using the long-chain DNA fragment-introduced chloroplast transformation vector constructed in the present invention, it is also possible to introduce a series of genes related to metabolic pathways and useful multiplexed genes into the chloroplast genome at once. Thus, it becomes possible to create new plants with new metabolic pathways and new functions.
Moreover, in the transformed plant of the present invention, since the introduced long gene to be introduced is directly introduced into the chloroplast genome instead of the nuclear genome, there is no fear that the introduced gene is diffused by pollen. That is, there is no concern about environmental pollution such as pollen being scattered over a wide area by wind or insects, such as a plant having a gene introduced into the nucleus. Moreover, the expression is stable between strains.
According to the transformation method of the present invention, by introducing a long chain (or multiple) gene encoding a protein having a beneficial function, the introduced gene is expressed in the transformed plant and has a beneficial function. The protein it has can be produced.

Furthermore, the prepared chloroplast transformation vector can also be used to construct a long-chain genomic library for genome function analysis, and clones having useful functions can be selected from the library and transformed directly into the chloroplast genome. .
Chloroplasts are thought to be organelles similar to or similar to prokaryotes such as cyanobacteria. In addition, chloroplast genome genes are known to be polycistronically expressed like prokaryotes. For this reason, a prokaryotic cyanobacterial genome library is prepared in Escherichia coli using a transformation vector for introducing long-chain DNA, thereby determining the sequence of the cyanobacterial genome and annotating the gene. Thus, if a clone containing a useful gene (group) is selected, the DNA of the selected clone can be directly introduced into the chloroplast genome. In addition, clones having a function can be selected by performing a specific functional screening from the prepared cyanobacteria genome library, and the DNA of the selected clone can be directly introduced into the chloroplast genome. For example, if a clone that fixes nitrogen is found from a library, the DNA of the clone can be introduced into the chloroplast genome.

  The chloroplast transformation vector of the present invention is characterized by having a useful long-chain DNA fragment of about 8 kbp or more introduced between two types of chloroplast genomic DNA. The chloroplast transformation vector of the present invention is described in detail below.

  In the present invention, “chloroplast” refers to a cell organelle in a leaf or other green tissue of a green plant. The green plant is a generic name for green plants, and any kind of plant can be used as long as it has chlorophyll and can perform photosynthesis. The green plants belong to the gates of green algae, axle algae, moss, ferns and seed plants. Preferable plants include tobacco, sorghum, Brassica, wheat, cotton, barley, sunflower, cucumber, lettuce, alfalfa, soybean, sorghum, poplar, rice or Arabidopsis thaliana, and tobacco is particularly preferable. In the present invention, the term “gene” includes not only DNA but also mRNA and cDNA thereof. Accordingly, the genes of the present invention include all of these DNAs, mRNAs and cDNAs.

  Examples of the chloroplast genomic DNA include chloroplast genomic DNA of various plants that have already been registered and clarified in Genebank, for example, tobacco chloroplast genomic DNA (accession No. Z00044 S54304).

  The “two types of DNA derived from chloroplast genomic DNA” may be any DNA having a base sequence capable of homologous recombination with chloroplast genomic DNA, other than structural genes and structural genes present in chloroplast genomic DNA. In the chloroplast genomic DNA, in the base sequence of the region where two types of structural genes are connected across the non-coding region, the non-coding region Arbitrary position of the region (position of at least about 50 bp, preferably about 100 to 1000 bp, more preferably about 200 to 500 bp from the 5 ′ end to the 3 ′ side or from the 3 ′ end to the 5 ′ side of the non-coding region) Each having two types of base sequences obtained by dividing into two (hereinafter referred to as structural gene regions for structural genes linked to the non-coding region). The type of DNA is preferred. The base sequence of the structural gene region need not have all base sequences obtained by dividing. The base sequence has at least about 700 bp, preferably about 700 to 2000 bp from the position divided by the non-coding region to the structural gene side, and at least the base sequence of the structural gene linked to the divided non-coding region. About 50 bp (preferably about 100 to 1000 bp, more preferably about 200 to 800 bp)

The “structural gene” refers to a region encoding protein, ribosomal RNA, or transfer RNA in chloroplast genomic DNA. Preferred examples of the structural gene include an rbcL gene encoding a large subunit of Rubisco, which is an enzyme involved in photosynthesis, and an accD gene encoding an acetyl CoA carboxylase beta subunit of an enzyme involved in fatty acid synthesis in plants. In addition, preferable structural genes derived from chloroplast genomic DNA other than the rbcL gene and the accD gene include trnG (tRNA-Gly (GCC)), trnfM (tRNA-fMet (CAU)), trnI (tRNA-Ile (GAU)). ), TrnA (tRNA-Ala (UGU)), trnV (tRNA-Val (GAC)) or 3′rps12 (ribosomal protein S12 exon-3) gene.
The “non-coding region” refers to a region other than a structural gene encoding a protein, ribosomal RNA, or transfer RNA on chloroplast genomic DNA. Non-coding regions include gene expression regulatory regions (eg, promoters, terminators, etc.) and the like.

  Preferred examples of the two types of structural genes in the region connected across the non-coding region include trnG and trnfM, rbcL gene and accD gene, trnI and trnA, or 3'rps12 gene and trnV. Particularly preferred are the rbcL gene and the accD gene.

  In the nucleotide sequence of the region where two types of structural genes are connected across the non-coding region on the chloroplast genome, the two types of structural gene regions obtained by dividing into two at any position of the non-coding region include trnG Examples include regions and trnfM regions; rbcL gene regions and accD gene regions; trnI regions and trnA regions; 3′rps12 gene regions and trnV regions. Preferably, the rbcL gene region and the accD gene region.

The DNA derived from chloroplast genomic DNA is substantially the same sequence as the DNA derived from chloroplast genomic DNA described above, or the DNA derived from chloroplast genomic DNA or the sequence substantially identical to the DNA. And DNA having a complementary base sequence. Moreover, DNA having a sequence showing homology with DNA derived from chloroplast genomic DNA and having a sequence capable of homologous recombination with chloroplast genomic DNA can be mentioned.
The term “substantially identical” refers to a single nucleotide polymorphism (SNP).
The “complementary base sequence” refers to a base sequence formed in chloroplast genomic DNA according to the base pairing rules (adenine / thymine, cytosine / guanine) with respect to the base sequence of the chloroplast genomic DNA. .
The “sequence showing homology” means, for example, the degree of coincidence with the nucleotide sequence of the structural gene region in chloroplast genomic DNA is at least about 50%, preferably about 60% or more, more preferably about 80% or more, More preferably, it refers to a base sequence that is about 90% or more, particularly preferably about 95% or more, or a base sequence that is not completely complementary but has sufficient base pairs to form a stable structure.

  In addition, the DNA derived from chloroplast genomic DNA is hybridized under stringent conditions with DNA having a base sequence complementary to the DNA derived from chloroplast genomic DNA described above, and chloroplast genomic DNA. The DNA used in the present invention can also be obtained by isolating DNA capable of homologous recombination with. “DNA that hybridizes under stringent conditions” means, for example, a colony hybridization method, a plaque hybridization method, or a Southern blot hybridization method using the partial sequence of DNA shown in SEQ ID NO: 1 as a probe. It means DNA obtained by use. Here, “stringent conditions” refer to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. Although it is difficult to quantify this condition clearly, DNAs having high homology, for example, at least about 50%, preferably about 60% or more, more preferably about 5% or more with the base sequence represented by SEQ ID NO: 1. A condition in which DNAs having a homology of 80% or more hybridize to each other and DNAs having lower homology to each other do not hybridize, or an SSC solution having a concentration of about 0.1 to 2 times (single-concentration SSC The composition of the solution consists of 150 mM sodium chloride and 15 mM sodium citrate.) Hybridization conditions at a temperature of about 65 ° C. The homology is calculated by BLAST.

The position where the long DNA fragment is introduced into the chloroplast genome varies depending on the DNA derived from the two types of chloroplast genome DNA used in the vector of the present invention. Two types of DNA derived from chloroplast genomic DNA are derived from trnG region-derived DNA and trnfM region-derived DNA, rbcL gene region-derived DNA and accD gene region-derived DNA, trnI region-derived DNA and trnA region-derived DNA, or 3'rps12 gene region In the case of DNA and trnV region-derived DNA, the long-chain DNA fragments are respectively located between the trnG region and trnfM region, between the rbcL gene region and the accD gene region, between the trnI region and the trnA region, or 3 ′. A non-coding region is desirable, such as between the rps12 gene region and the trnV region, and at a sufficient distance from each structural gene. The sufficient distance is at least about 50 bases from the structural gene, preferably about 100 to 1000 bases, more preferably about 200 to 500 bases.
The chloroplast transformation vector of the present invention using rbcL gene region-derived DNA and accD gene region-derived DNA as DNA derived from chloroplast genomic DNA will be described in more detail below. Examples of the base sequence of the region where the rbcL gene and the accD gene are connected via the non-coding region include, for example, accession No. registered in Genebank as tobacco chloroplast DNA. 570004 in Z00044 S54304. . The base sequence of the 61331 region can be mentioned.

The rbcL gene is a Rubisco large subunit gene encoded in the chloroplast genome. The large Rubisco subunit is a key enzyme that catalyzes the CO ₂ fixation reaction (carboxylase reaction), which is the initial stage, in the photosynthetic CO ₂ fixation reaction circuit (calvin cycle), and controls the rate of turnover in the circuit. The enzyme also catalyzes a reaction for fixing oxygen (O ₂ ) (oxygenase reaction).

  As the DNA derived from the rbcL gene region, for example, DNA having the base sequence of the rbcL gene region derived from tobacco chloroplast genomic DNA [base sequence (1729 bp) shown in SEQ ID NO: 1 of Sequence Listing] can be used. Further, the base sequence complementary to the base sequence of the rbcL gene region, or one or a plurality of bases in the base sequence of the rbcL gene region or the complementary base sequence thereof, for example, 1 to several (about 5 to about 5 10) DNA modified by deletion of one base, substitution by another base, or addition of one to several (about 5-10) other bases Any sequence capable of homologous recombination with the tobacco chloroplast rbcL gene region can be preferably used.

  In addition, any DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the rbcL gene region-derived DNA and can homologously recombine with the chloroplast rbcL gene is used in the present invention. “DNA that hybridizes under stringent conditions” and “stringent conditions” are the same as described above.

  The accD gene is a gene of acetyl CoA carboxylase beta subunit encoded by the chloroplast genome. Acetyl CoA carboxylase is an enzyme involved in fatty acid synthesis in plants.

  As the accD gene region-derived DNA, for example, DNA having the base sequence of the accD gene region derived from tobacco chloroplast genomic DNA [base sequence (1183 bp) shown in SEQ ID NO: 2 of the sequence listing]) can be used. Further, a base sequence complementary to the base sequence of the accD gene region, or one or a plurality of bases in the base sequence of the accD gene region or the complementary base sequence thereof, for example, 1 to several (about 5 to about 5 10) DNA modified by deletion of one base, substitution by another base, or addition of one to several (about 5-10) other bases Any sequence capable of homologous recombination with the tobacco chloroplast accD gene region can be preferably used.

  Any DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the accD gene region-derived DNA and can homologously recombine with the chloroplast accD gene is used in the present invention. “DNA that hybridizes under stringent conditions” and “stringent conditions” are the same as described above.

  In addition, when the chloroplast whole genome base sequence of the plant to be transformed is unknown, the chloroplast genome of the plant is known by a known method, for example, New Chemistry Experiment Course Volume 2 Nucleic Acid 1 Separation and Purification (Edited by the Japanese Biochemical Society) Tokyo Chemical Doujin, pp. 29-32 (1991), CTAB (Cetyl trimethyl ammonium bromide) method and the like, and can be isolated according to well-known techniques based on the sequence homology of the gene. In this case, all or part of a known coding sequence (for example, the base sequence of rbcL or accD gene of tobacco chloroplast genome) is compared to the sequence present in the plant-derived genomic library or cDNA library. Or as a probe that selectively hybridizes, or used to design PCR primers.

  The vector of the present invention comprises a DNA comprising a useful long-chain DNA fragment (hereinafter simply referred to as a long-chain DNA fragment) of about 8 kbp or more between the DNA sequences derived from the two types of chloroplast genomic DNA. Can be inserted. The above-mentioned "can insert a DNA containing a long-chain DNA fragment of about 8 kbp or more" means that a DNA containing a DNA fragment of less than about 8 kbp can naturally be inserted. This means that DNA containing a long DNA fragment can also be inserted.

  The long DNA fragment is a DNA fragment having a base sequence of about 8 kbp or more, preferably about 8 to 300 kbp, more preferably about 8 to 200 kbp, more preferably about 8 to 120 kbp, and particularly preferably about 8 to 80 kbp. The type of the DNA is not particularly limited as long as the DNA exhibits a useful function when expressed. Examples of DNA that exhibits useful functions include DNA fragments containing genes that improve plant material productivity, DNA fragments containing genes that improve environmental resistance, or plants that are energy-saving or low environmental load industrial raw materials Examples thereof include DNA fragments containing a plurality of genes (including regulatory genes) necessary for use as a production process. The long-chain DNA fragment may be a plant-specific gene or a foreign gene (for example, a gene of other species of plant, animal, bacteria, yeast, etc.). The long-chain DNA fragment may be a DNA fragment in which one gene has a base sequence of about 8 kbp or more, or a DNA fragment in which a plurality of genes are linked to form a base sequence of about 8 kbp or more. Examples of such a long-chain DNA fragment include (a) a DNA fragment containing a fatty acid synthase gene (for example, fabH, fabD, fabG, acpP, fabF gene or a gene group thereof); (b) a nitrogen-fixing enzyme nitrogenase A DNA fragment containing a structural gene (for example, nitrogen-fixed nifH, nifD, nifK or a gene group thereof, nifA of a nitrogen-fixing enzyme nitrogenase control gene); (c) constitutes a rate-limiting enzyme group of the Calvin cycle involved in photosynthesis A DNA fragment linking DNA encoding an enzyme; (d) a DNA fragment containing an enzyme gene (for example, carotenoid biosynthetic enzyme group, etc.) involved in biosynthesis of useful components produced in a plant; (e) disease-resistant protein DN containing a gene encoding (for example, Bacillus thuringes toxin etc.) A fragment; (f) a DNA fragment containing a gene encoding a herbicide-resistant protein (for example, 5-enolpyruvylshikimate-3-phosphate synthase); (g) an environmental stress resistance gene or a gene related to production efficiency (H) a DNA fragment containing a group of genes that biosynthesizes rubber; (i) a DNA fragment containing a group of genes that biosynthesizes a hydrocarbon; (j) DNA fragments containing genes that biosynthesize amino acids; (k) DNA fragments containing genes that biosynthesize biodegradable plastic raw materials, etc .; (l) cellulose synthase genes (eg, bcsA, bcsB, bcsC, bcsD, etc.) A DNA fragment comprising a group; (m) a DN comprising a polyketide compound synthase gene (eg, aveA1, aveA2, aveA3, etc.) group Fragment; (n) a DNA fragment containing the cellulolytic enzyme genes; DNA fragment such as a DNA fragment in combination of two or more represented by or from said (a) (n) and the like.

  Further, the chloroplast transformation vector of the present invention may have a ribosome binding site inserted upstream of the translation initiation point of DNA containing a long-chain DNA fragment. By placing a ribosome binding site upstream of the DNA containing the transgene, a protein based on a long DNA fragment can be highly expressed. The ribosome binding site may be upstream of the translation start point of the long DNA fragment, but is preferably about 7 to 11 bases upstream of the translation start point, and about 9 bases upstream. Is more preferable. Such a ribosome binding site may have a base sequence known per se that is known to be capable of binding to a ribosome, but an SD sequence is preferred. The SD sequence is an abbreviation for Shine-Dalgarno sequence and is a segment composed of about 4 to 7 nucleotides, and its base sequence is a part or all of 5'-AGGAGGU-3 '.

  In the chloroplast transformation vector of the present invention, a plant cell-derived promoter may be inserted upstream of the N-terminal side of DNA containing a long-chain DNA fragment. Examples of the promoter include an elongation factor 1α gene promoter (EF1α promoter), 35S promoter, psbA promoter, PPDK promoter, PsPAL1 promoter, PAL promoter, UBIZM1 ubiquitin promoter, rrn promoter, and the like. Among them, a promoter derived from chloroplast genomic DNA is preferable. When the ribosome binding site is inserted upstream of the translation start site of DNA containing a long-chain DNA fragment, the promoter is preferably inserted further upstream of the ribosome binding site. In this case, as long as the promoter is upstream of the ribosome binding site, it may be continuous with the ribosome binding site or may be about 1 to 30 bases upstream.

  In the chloroplast transformation vector of the present invention, a plant-derived terminator may be inserted downstream of the C-terminal side of DNA containing a long-chain DNA fragment. The terminator may be continuous with the DNA as long as it is downstream of the DNA containing the long-chain DNA fragment, or may be about 1 to 30 bases downstream. Examples of the terminator include 35S terminator, rps16 terminator, CaMV35S terminator, ORF25polyA transcription terminator, PsbA terminator and the like. Of these, terminators derived from chloroplast genomic DNA are preferred.

  In addition, the chloroplast transformation vector of the present invention preferably has a gene for identifying a gene recombinant. The gene for identifying the gene recombinant is not particularly limited, and a gene known per se may be used. Examples thereof include various drug resistance genes (aadA) or genes that complement the auxotrophy of the host. More specifically, for example, ampicillin resistance gene, neomycin resistance gene (G418 resistance), chloramphenicol resistance gene, kanamycin resistance gene, spectinomycin resistance gene, URA3 gene and the like can be mentioned. Further, a promoter for recognizing the gene (hereinafter abbreviated as aadA promoter) and a terminator for the gene (hereinafter abbreviated as aadA terminator) are arranged upstream and downstream of the gene, respectively. preferable. As the aadA promoter and aadA terminator, the above plant-derived promoters and terminators can be preferably used, but the rrn promoter and psbA terminator are particularly suitable. The aadA promoter / aadA / aadA terminator is sometimes referred to as an aadA cassette.

  Since the chloroplast transformation vector of the present invention has DNA derived from chloroplast genomic DNA, there is an advantage that long-chain DNA fragments are easily incorporated into the chloroplast genome of plants by homologous recombination.

  As a basic vector used for the chloroplast transformation vector of the present invention, a commercially available vector or an artificial chromosome which is a vector capable of cloning long-chain DNA can be used. Specific examples include yeast artificial chromosome (YAC) vectors (Molec. Reprod. Dev. 1997, 47, 158-163), bacterial artificial synthetic vector (BAC) vectors, fosmids, and the like. It is done. Examples of YAC vectors include pYAC2, pYAC3, pYAC4, and pYACNeo. Examples of the BAC vector include pCC1BAC, pBAC, plndigoBAC, and pBeloBAC. Examples of the fosmid include pCCFOS. BAC vectors are preferred.

  The vector of the present invention can be prepared, for example, according to the procedure shown in FIG. First, pUC19 in which a multicloning site is inserted into a region between rbcL gene region-derived DNA and accD gene region-derived DNA of tobacco chloroplast genomic DNA was prepared according to the method described in, for example, JP-A-2002-272476. Amplify. The multicloning site preferably has a plurality (about 2 to 10) of restriction enzyme sites (hereinafter also referred to as restriction enzyme sites). Examples of restriction enzyme sites include NotI site, NheI site, AscI site, PmlI site, ClaI site, MluI site, and SalI site. These restriction enzyme sites can be cleaved with restriction enzymes NotI, NheI, AscI, PmlI, ClaI, MluI or SalI, respectively.

  Next, a region containing a multicloning site is cut out from the amplified pUC19 in a region between the rbcL gene region-derived DNA and the accD gene region-derived DNA using a restriction enzyme (for example, EcoRI and HindIII). The cut out region is inserted into a BAC vector, for example, pCC1BAC (EPICENTER) by a known method to prepare pTA05. Next, the chloroplast transformation of the present invention is performed by cleaving an arbitrary restriction enzyme site of the multicloning site of the region inserted into pTA05 with a specific restriction enzyme and inserting an aadA cassette into the cleavage site. Vector pTA500 can be prepared. In addition, for example, long DNA fragments can be similarly introduced into other restriction enzyme sites of the multicloning site.

  The vector pTA500 of the present invention thus produced is illustrated as a schematic diagram in FIG. 2 as one embodiment of the present invention. In the figure, rbcL represents the rbcL gene region derived from tobacco chloroplast genomic DNA. accD represents the accD gene region derived from tobacco chloroplast genomic DNA. Most of the bold lines represent pCC1BAC-derived base sequences. Prrn represents the rrn promoter derived from tobacco chloroplast genomic DNA. aadA represents a spectinomycin resistance gene. TpsbA represents a psbA terminator derived from tobacco chloroplast genomic DNA. The site describing the base sequence is a multicloning site. This means that long-chain DNA is inserted into at least one of the restriction enzyme sites of the multicloning site, preferably the AscI site, PmlI site, ClaI site, MluI site, and SalI site.

  The chloroplast transformation vector thus prepared is introduced into a host cell to produce a transformant. In this case, the host cell is preferably a plant cell, and more preferably a plant chloroplast. As a method for transforming by introducing the chloroplast transformation vector of the present invention into a host cell, particularly its chloroplast, known methods such as the particle gun method (Svab, Z., Hajdukiwicz, P., and and others) Maliga, P., Proc. Natl. Acad. Sci. USA, 1990, Vol. 87, p. 8526-8530) or the PEG method (Golds, T., Maliga, P., and Koop, H.-U. Bio / Technol., 1993, Vol. 11, p. 95-97) and the like can be preferably used. For example, in the particle gun method, a vector can be introduced into a host cell by coating the vector with very fine particles of gold or tungsten, and driving the particle to which the vector is adhered into the host cell with an explosive or high-pressure gas.

  The above-mentioned genetic engineering or biotechnological operations are described in commercial experiments, for example, Molecular Cloning, Cold Spring Harbor Laboratory published in 1982, published in 1989. It can be easily performed according to the method described in Molecular Cloning, 2nd edition (Molecular Cloning, 2nd ed.) Cold Spring Harbor Laboratory, etc.

Plant leaf cells into which the vector has been introduced can be cultured in a medium and redifferentiated into a plant body. Examples of the basal medium for the callus induction medium or the regeneration differentiation medium include Gamborg B5, Murashige-Skoog (MS), and Niche & Nitch. It is preferable to add a support for solidifying the medium to the medium. Examples of the support include agar, gellan gum, paper bridge and the like.
Although the density | concentration of a support body changes with pH etc. of a culture medium, in the case of gellan gum, for example, about 0.1-0.5 mass%, More preferably, about 0.15-0.25 mass% is preferable.

  If desired, plant hormones can be added to the basal medium. Examples of plant hormones include auxin (eg, naphthalene acetic acid, 2-naphthoxyacetic acid, indole acetic acid, 4-chloroindole acetic acid, indole butyric acid or 2,4-dichlorophenoxyacetic acid) or cytokinin (eg, kinetin, zeatin, benzyladenine, Isopentenyl adenine, 4-pyridylphenyl urea, etc.). The concentration of auxin and cytokinin varies depending on the auxin and cytokinin used, but can be added to the basal medium to be about 0.01 to 1.0 mg / L, preferably about 0.05 to 0.2 μM.

In addition, amino acids (eg, glycine, etc.), adenine and / or coconut water may be added to the basal medium.
The medium is usually adjusted to about pH 4-8, preferably about pH 5-7 with potassium hydroxide or the like.

  The plant transformed by the present invention may be of any kind, such as tobacco, sorghum, Brassica, wheat, cotton, barley, sunflower, cucumber, lettuce, alfalfa, soybean, sorghum, poplar, rice or Arabidopsis. Can be mentioned. Tobacco is preferred.

  The plant transformed by the present invention expresses a gene contained in the introduced long DNA fragment, and can impart various functions to the plant. For example, by introducing genes that encode enzymes involved in photosynthesis, which play an important role in carbon dioxide fixation, into plants, the production of enzymes involved in photosynthesis is higher than that of normal plants. Certain photosynthesis functions are improved. As a result, the plant grows early and the yield of the plant increases. In addition, since a plant into which the gene group has been introduced and production of an enzyme involved in photosynthesis is enhanced can increase the carbon dioxide uptake rate in the atmosphere, the concentration of carbon dioxide in the atmosphere can be increased by cultivation of the plant. Can be reduced. Therefore, cultivating the plant can contribute to the suppression of global warming. By introducing a gene encoding a nitrogen-fixing enzyme, the plant expressing the transformed gene has a higher nitrogen-fixing enzyme than normal plants, and can grow under low nitrogen conditions; Reduction of chemical nitrogen fertilizer etc. can be achieved, and environmentally friendly plant production becomes possible. In addition, it is possible to produce plants that can withstand the depletion of nitrogen sources in mountains and the like where it is difficult to artificially apply fertilizer. By introducing a gene encoding a fatty acid synthase, the plant expressing the transformed gene has higher fatty acid synthase than a normal plant, promotes fatty acid synthesis in the plant, and increases the production of vegetable oil. be able to. In addition, since an enzyme (for example, carotenoid biosynthetic enzyme group) involved in biosynthesis of useful components produced in a plant body can be expressed, a plant having a high content of useful components such as carotenoids can be produced. Pest-resistant proteins such as Bacillus thuringes toxin, herbicide-resistant proteins such as 5-enolpyruvylshikimate-3-phosphate synthase, or proteins such as catalase related to environmental stress resistance are produced in plants This makes it possible to produce plants that are resistant to pests, plants that are resistant to herbicides, plants that are highly adaptable to the environment, and the like. In addition, rubber, hydrocarbons, amino acids, biodegradable plastic raw materials, cellulose, polyketide compounds and / or cellulolytic enzymes can be synthesized using plants.

In addition, the chloroplast transformation vector of the present invention can be used to produce a genomic DNA library. Examples of the library include cyanobacteria genomic DNA library.
A genomic DNA library such as cyanobacteria can be prepared by the following method.
(1) First, cyanobacteria genomic DNA is extracted. Cyanobacterial genomic DNA can be obtained from, for example, Shinsei Chemistry Laboratory Volume 2 Nucleic Acid 1 Separation and Purification (Edited by the Japanese Biochemical Society), Tokyo Chemical Dojin, pp. 29-32 (1991).
(2) Next, the restriction enzyme site of the multicloning site of the chloroplast transformation vector inserted between the two chloroplast genome DNAs before introduction of the long DNA fragment is cleaved with a restriction enzyme. To do. The restriction enzyme can be implemented using one or two restriction enzymes that can cleave the restriction enzyme site of the multicloning site. For example, the MluI site may be cleaved with the restriction enzyme MluI. The cleavage end of the restriction enzyme is preferably dephosphorylated. The dephosphorylation treatment can be performed using, for example, commercially available alkaline phosphatase (BAP, CIAP, SAP: Takara Bio Inc.).
(3) The cyanobacterial genomic DNA extracted in the above (1) is digested with the same restriction enzyme as that obtained by cleaving the multicloning site in the above (2), for example, MluI.
(4) The cyanobacterial genomic DNA digested with the restriction enzyme is inserted by ligation into the cleavage site of the multicloning site of the chloroplast transformation vector. Ligation can be performed using, for example, Fast-Link DNA Ligase (EPICENTER).
(5) A cyanobacterium genomic DNA library can be prepared by introducing a vector into which a cyanobacterium genomic DNA digested with a restriction enzyme is inserted into a host such as Escherichia coli. Introduction into the host can be carried out by a per se known method such as electroporation.

  Hereinafter, preparation of a vector for introducing long-chain DNA fragment of the present invention into a chloroplast transformation vector and an example of using this to introduce a cyanobacterial genomic DNA fragment into tobacco chloroplast will be described. The present invention is not limited to the examples.

Construction of Long DNA Fragment-Introduced Chloroplast Transformation Vector The base sequence of the long DNA fragment-introduced chloroplast transformation vector is shown in SEQ ID NO: 3 in the Sequence Listing. First, a multicloning site (SEQ ID NO: 4 in the sequence listing) is placed in a region between the rbcL gene region-derived DNA (SEQ ID NO: 1 in the sequence listing) and the accD gene region-derived DNA (SEQ ID NO: 2 in the sequence listing) of the tobacco chloroplast genome. ) Was inserted according to the method described in JP-A-2002-272476. The multicloning site has NheI site, AscI site, MluI site, PstI site, NotI site, SalI site, PmlI site and ClaI site as restriction enzyme sites.
A region containing rbcL gene region-derived DNA / multicloning site / accD gene region-derived DNA was excised from the amplified pUC19 using EcoRI and HindIII restriction enzymes. Next, the excised region was inserted into the EcoRI site and HindIII site of pCC1BAC (EPICENTER) to prepare pTA05.
Separately, the aadA cassette was prepared by using pLD6 (accession NO. BD174931) as a template as a forward primer (SEQ ID NO: 5; 5′-GCTAGCTCTAGTGTGATTTGCTCCCC-3 ′) and reverse primer (SEQ ID NO: 6; 5′-GGCGGCGCTCTCGAATATAGCTCTTCTCTC) 3 ') and amplified by PCR and introduced into the NheI and AscI sites of the multicloning site of pTA05. The 5 ′ end of the forward primer has an NheI restriction enzyme recognition site, and the 5 ′ end of the reverse primer has an AscI restriction enzyme recognition site. In this manner, a basic chloroplast transformation vector pTA500 (FIG. 2; SEQ ID NO: 3 in the sequence listing) for introducing a long DNA fragment of the present invention was prepared. In the nucleotide sequence of SEQ ID NO: 3 in the sequence listing, the DNA derived from the rbcL gene region is 333 to 2061, the aadA cassette is 2079 to 3418, and the accD gene region DNA is 3453 to 4635. Corresponds to the second.

Preparation of cyanobacterial genomic DNA library using long-chain DNA fragment-introduced chloroplast transformation vector (1) Preparation of cyanobacterial genomic DNA fragment The cyanobacteria (Cyanothes sp. TU126) is an A-N without a nitrogen source. Culture was performed at 27 ° C. for about one month using a medium (Mitsui et al., Nature, 1988, 323, p. 720-722). Genome extraction was performed according to the following procedure. Cells having a wet weight of about 600 mg were suspended in a washing solution [50 mM Tris-HCl, 1 mM EDTA (pH 8.0)] and washed. 1.5 mL of saturated sodium iodide aqueous solution was added to the cells, suspended using a mixer, and kept warm (37 ° C., 20 minutes). The cells were washed with sterilized water and suspended in 540 μL of TEN buffer [50 mM Tris-HCl, 20 mM EDTA (pH 8.0), 100 mM NaCl]. 60 μL of 50 mg / mL lysozyme was added, mixed, and incubated (37 ° C., 20 minutes). Furthermore, after adding 20 mg / mL protease K solution (Wako Pure Chemical Industries, Ltd.), 30 μL of 10% SDS (sodium dodecyl sulfate) was added and incubated (55 ° C., overnight). An equal amount of TE saturated phenol (Nippon Gene Co., Ltd.) was added and gently mixed (room temperature, 20 minutes) (hereinafter referred to as phenol treatment). Centrifugation was performed, the aqueous layer was recovered, and phenol treatment was performed again. After centrifugation, an equal volume of chloroform-isoamyl alcohol [24: 1 (V / V)] was added to the recovered aqueous layer and mixed gently. Centrifugation was performed, and the aqueous layer was recovered. Chloroform-isoamyl alcohol [24: 1 (V / V)] extraction was performed until the intermediate layer disappeared. An equal amount of diethyl ether was added to the recovered aqueous layer and centrifuged to recover the aqueous layer. Diethyl ether treatment was performed again. 2.5 volumes of ethanol was added to the aqueous layer, mixed gently, and then centrifuged. The supernatant was discarded, the pellet was washed with 70% ethanol, and then dissolved in 100 μL of TE buffer (50 mM Tris-HCl, 1 mM EDTA (pH 8.0)). The cyanobacterial genome thus extracted was added with 10 units of MluI to 2 μg of the genome and reacted at 37 ° C. overnight to prepare a cyanobacterial genomic DNA fragment.

(2) Preparation of cyanobacterial genomic library The MluI site of the multicloning site of the chloroplast transformation vector pTA500 for introducing long DNA fragments prepared in Example 1 was cleaved with a restriction enzyme, and the end was dephosphorylated. did. Dephosphorylation was performed according to the attached protocol using alkaline phosphatase [Cal intestine alkaline phosphatase (CIAP); Takara Bio Inc.]. Ligation of the phosphorylated long-chain DNA-introduced chloroplast transformation vector pTA500 and the cyanobacterial genomic DNA fragment prepared in (1) above was performed using Fast-Link DNA Ligase (EPICENTER) according to the attached protocol. It was. A genome library was prepared by introducing the above-mentioned vector ligated with cyanobacterial genomic DNA fragment into E. coli by electroporation.
Colonies were randomly selected from the prepared library and cultured in an LB (Luria-Bertani) medium supplemented with spectinomycin and chloramphenicol at 37 ° C. for about 16 hours. The plasmid was purified from the cultured E. coli using QIAprep Spin Miniprep kit (QIAGEN). The purified plasmid was treated with MluI (MluI, 1 unit for about 1 μg of plasmid) (37 ° C., 2 hours), and pulsed field electrophoresis (electrophoresis time 13 hours 45 minutes, forward 9 V / cm 2, reverse 6 V / cm, 0 .05-0.92 sec), the size of the cyanobacterial genome introduced into the long-chain DNA chloroplast transformation vector pTA500 was examined. Attempts were made to introduce three types of genomes of about 23, 34, and 35 kbp into the tobacco chloroplast genome.
At the same time, these three types of clones were sequenced. The sequence reaction was performed using CEQ DTCS Quick Start kit (BECKMAN), and the sequence was performed with CEQ8000 system (BECKMAN). Each method followed the attached protocol. As for the primer for the sequence, the upstream side is 5′-GAACTTGTTTCTCTTCTTGC-3 ′ (forward primer; SEQ ID NO: 21 in the sequence listing; corresponding to the 3263-3282nd position in SEQ ID NO: 3 in the sequence listing), and the downstream side is 5′-CAACACGGGAACAAAGGGGAC -3 ′ (reverse primer; SEQ ID NO: 12 in the sequence listing; equivalent to 3549-3568 in SEQ ID NO: 3 in the sequence listing) was used.

The long-chain DNA fragment-introduced chloroplast transformation vector pTA500, into which the cyanobacterial genomic DNA fragment obtained in Example 2 was inserted, was used as a particle gun (type PDS-1000 / He BIO-RAD) on the chloroplast of tobacco leaf cells. To produce a chloroplast-transformed plant. Particle gun introduction method and differentiation induction from tobacco leaf cells into which a vector has been introduced into a plant are known methods [Zora Svab, Peter Hajdukiwicz, and Pal Malliga, Proc. NatlAcad. Sci. USA, 87, 8526-8530 (1990)]. That is, 2.3 mg of gold particles having a diameter of 0.6 microns, 25 μg of the chloroplast transformation vector prepared in Example 2, 250 μL of 2.5 M calcium chloride, and 50 μL of 0.1 M spermidine were mixed every minute. Turbidity was carried out 10 times (still in ice between suspensions) to attach the vector to the gold particles. The gold particles were washed twice with ethanol and then suspended in 60 μL of ethanol. This corresponds to an amount that can be used for 10 driving experiments. Gold particles were implanted into tobacco leaf cells using a particle gun (model PDS-1000 / He BIO-RAD). Ten implants were performed per experiment. The driving pressure was 900 psi. Two days after the implantation, the fresh leaves implanted with the vector were cut into 3 mm square sections with a scalpel and placed on MS (Murashige & Skog) medium containing spectinomycin (50 mg / L).
FIG. 4 shows a plant body which has been differentiated into a plant from a tobacco leaf cell having about 23 kbp of cyanobacterial genome fragment introduced into the chloroplast genome of the tobacco leaf cell.

Analysis of Tobacco Chloroplast Transformant by PCR About 50 mg of tobacco plant leaf tissue obtained in Example 3 was collected and crushed under liquid nitrogen. Add the DNA extraction buffer [0.3 M sodium chloride, 0.05 M and squirrel hydrochloric acid (pH 7.5), 20 mM EDTA, 0.5 mass% sodium dodecyl sulfate, 5 M urea, 5 mass% phenol] to the crushed sample, Mixed. Next, the mixture was extracted with phenol / chloroform / isoamyl alcohol [25/24/1 (V / V / V)], DNA was precipitated with ethanol, and the extracted DNA was air-dried. To the dried extracted DNA, 100 μL of TE solution (10 mM Tris-HCl, pH 8.0; 1 mM EDTA, pH 8.0) was added to dissolve the DNA. In PCR for confirming the presence or absence of introduction of cyanobacterial genomic DNA fragments, as shown in Table 1, three kinds of forward primers (LT, MT and ST in Table 1) were used as a template with a DNA solution extracted from tobacco plants as a template. And reverse primer (LB, MB, SB in Table 1) set were used for PCR reaction.

The results of analyzing the PCR product by agarose gel electrophoresis are shown in FIG. A 23 kbp band was observed in the DNA extract of tobacco leaves (Fig. 5-2; A) into which the 23 kbp DNA fragment of Example 3 was inserted and the vector used in Example 3 (Fig. 5-2; C). It was. The base sequence of the 23 kbp DNA fragment is shown in SEQ ID NO: 22 in the sequence listing. This indicates that the chloroplast transformation vector of the present invention can insert a DNA fragment of 23 kbp (about 8 kbp or more).
PCR was performed in the same manner using the primers of Table 2, and a 34 kbp DNA fragment (SEQ ID NO: 23 in the sequence listing) and a 35 kbp DNA fragment (SEQ ID NO: 24 in the sequence listing) were confirmed.

  When the vector of the present invention is used, a long-chain DNA fragment can be introduced into the chloroplast genome of a plant, which is useful for the creation of a new plant having a new metabolic pathway and function.

FIG. 1 is a diagram showing an embodiment of a process for constructing a vector for transforming a long DNA fragment chloroplast. FIG. 2 is a schematic diagram of a long-chain DNA fragment chloroplast transformation vector pTA500. rbcL represents the rbcL gene region derived from tobacco chloroplast genomic DNA. accD represents the accD gene region derived from tobacco chloroplast genomic DNA. Most of the bold lines represent pCC1BAC-derived base sequences. PmlI, ClaI, MluI, and SalI represent restriction enzyme sites. Prrn represents the rrn promoter derived from tobacco chloroplast genomic DNA. aadA represents a spectinomycin resistance gene. TpsbA represents a psbA terminator derived from tobacco chloroplast genomic DNA. PpsbA represents the psbA promoter derived from tobacco chloroplast genomic DNA. The site describing the base sequence is a multicloning site. FIG. 3 shows the results of pulse field electrophoresis of a fragment obtained by cleaving a cyanobacterium MluI complete digested genomic library prepared with a long DNA fragment chloroplast transformation vector with the restriction enzyme MluI. FIG. 4 is a photograph of a plant in which a cyanobacterial genome fragment of about 23 kbp was introduced into the tobacco chloroplast genome. FIG. 5 shows a schematic diagram (FIG. 5-1) and agarose electrophoresis (FIG. 5-2) of the positions of PCR primers examined for introduction of about 23 kbp of cyanobacterial genome fragment into the tobacco chloroplast genome. In the figure, LT, LB, MT, MB, ST and SB are the names of the primers used. PCR was performed with LT / LB, MT / MB, and ST / SB primer sets. 1.8, 23, and 0.5 kbp represent the amplification size. A, B, and C represent PCR templates. A: Tobacco transformant DNA extract, B: Tobacco wild type DNA extract, C: Transfer plasmid, M represents a marker. M1: λ / HindIII, M2: 1 kbp ladder

Claims (9)

(a) It has a region where two types of structural genes on the chloroplast-derived genome are connected across the non-coding region ,
(b) having a multi-cloning site represented by SEQ ID NO: 4 in the non-coding region,
(c) having a non-coding region of 50 to 1000 bp at both ends of the multicloning site;
(d) 8-80 kbp of useful long DNA fragment to be introduced is introduced into the multicloning site,
(e) DNA sequences of the respective structural genes are separated by the multiple cloning site, and, a DNA sequence of non-coding region adjacent thereto is 700～2000Bp,
(f) the two types of structural genes, r Bcl gene and accD long DNA fragment introduced chloroplast transformation vector, which is a gene.   The vector according to claim 1, wherein the long DNA fragment is a DNA fragment derived from cyanobacterial genomic DNA.   A cyanobacterial genome library prepared with the vector according to claim 2.   A plant transformed with the vector according to claim 1.   A method for transforming a plant chloroplast, comprising introducing the vector according to claim 1 or 2 into a plant chloroplast.   6. The transformation method according to claim 5, wherein the introduction of the vector uses a particle gun. (a) It has a region where two types of structural genes on the chloroplast-derived genome are connected across the non-coding region,
(b) having a multi-cloning site represented by SEQ ID NO: 4 in the non-coding region,
(c) having a non-coding region of 50 to 1000 bp at both ends of the multicloning site;
(d) A useful long DNA fragment of 8 to 80 kbp can be introduced into the multicloning site,
(e) The DNA sequence of each structural gene divided at the multicloning site and the DNA sequence of the non-coding region adjacent thereto are 700 to 2000 bp,
(f) The two types of structural genes are the rbcL gene and the accD gene
A long-chain DNA fragment-introduced chloroplast transformation vector characterized by the above. 8. The vector according to claim 7 , wherein the long DNA fragment-introduced chloroplast transformation vector has a base sequence of SEQ ID NO: 3. The vector according to any one of claims 1, 2 and 7 , wherein the long-chain DNA fragment-introduced chloroplast transformation vector is a BAC vector.