JP4740567B2 - A method for transformation of chloroplasts of Asteraceae plants - Google Patents

Description

  The present invention relates to a method for transforming chloroplasts of Asteraceae plants.

  Currently, with regard to industrial protein production methods, research has already been conducted on the production of medical proteins by introducing genes that express proteins useful as pharmaceuticals into the nuclear chromosomes of Escherichia coli and plants. Is progressing in each country. However, when bacteria such as E. coli are used, the toxin cannot be prevented from being mixed into the final product. In addition, protein production methods have been developed using transformed plants that have been introduced into the nuclear genome of the plant, but problems remain, such as low protein production. In addition, in the case of introducing a gene into the nucleus of a plant, the propagation of the gene to nature due to pollen scattering is regarded as a problem. Under such circumstances, a method for transformation into a plant chloroplast genome was put into practical use in tobacco by Mariga et al. A major feature of chloroplast transformation is that chloroplasts have the ability to produce and accumulate a large amount of protein as compared to transformation into the nuclear genome of plants. Other characteristics of chloroplast transformation are that the genetic information of chloroplasts is generally maternally inherited, taking advantage of the low possibility of horizontal transmission of transgenes through pollen and the polycistronic control of chloroplasts. Advantages such as multigene expression. Because of these advantages, transformed chloroplasts are beginning to attract attention as a useful system for producing substances such as proteins. For example, a vector capable of highly expressing a target protein in tobacco chloroplast is known. This vector is characterized by having a psbA promoter and a ribosome binding site upstream of the translation start point of the gene encoding the protein (see Patent Document 1). The purpose of this method is to produce a protein having pharmacological activity, a protein useful as a material for the pharmaceutical industry, etc. using tobacco instead of being produced by microorganisms. However, this method is not suitable for protein production that requires advanced purification for medical use because the plant to be transformed is tobacco, and it also produces substances harmful to the body as secondary metabolites of tobacco. It can be said that.

As the transformation of Asteraceae plants, for example, lettuce big bain virus resistant lettuce (see Patent Document 1) obtained by introducing DNA that suppresses the production and function of lettuce big bain virus protein into lettuce cells. Known is lettuce transformed with a plant transformation vector containing a gene of a lettuce infectious yellowing virus protein or a part of the gene (Patent Document 2). However, in any of these transformed lettuce, each gene is introduced into the leaf nuclear genome by introducing a plasmid constructed using the gene into Agrobacterium and infecting the leaf disk. The gene was not directly introduced into the chloroplast.
JP 2002-272476 A International Publication No. 01/090362 Pamphlet International Publication No. 95/02056 Pamphlet Zora Svab and two others, Proceeding of the National Academy of Science of the United State of America (Proc. Natl Acad. Sci. USA), 1990 87, p. 8526-8530

  An object of the present invention is to provide a transformation method capable of efficiently expressing a useful protein such as a medical protein in a chloroplast of an Asteraceae plant without producing harmful secondary metabolites.

  The present inventors have focused on the Asteraceae lettuce as a plant that grows quickly and has a low possibility of producing harmful secondary metabolites. Since lettuce can also be cultivated in a plant factory using hydroponics, we tried to construct a stable protein production system that is not affected by weather or pest damage using lettuce. In view of the above, the present inventors have conducted intensive research on protein production methods in lettuce chloroplasts. First, the base sequence of lettuce chloroplasts was determined in order to obtain the base sequence derived from lettuce chloroplasts and to examine the position where the foreign gene was introduced. Homologous sequences that enable efficient homologous recombination with the lettuce chloroplast genome from the determined base sequence of lettuce chloroplasts (ribulose-1,5-biscarboxylase / oxygenase large subunit gene and acetyl CoA carboxylase subunit gene) And a vector having a lettuce chloroplast genome-derived expression regulatory sequence (rRNA operon promoter and psbA gene terminator) for surely expressing the transgene in lettuce chloroplasts. The present inventors have also studied the method of gene transfer into the lettuce chloroplast genome using the constructed vector. In addition, the present inventors also paid attention to the importance of the step of regenerating lettuce leaf cells into which the gene has been introduced into the lettuce chloroplast genome into plant individuals, and lettuce that has introduced the gene into the lettuce chloroplast genome. The present inventors completed the present invention by studying the redifferentiation conditions of leaf slices and further examining the cultivation method of plant solids thus obtained.

That is, the present invention
(1) A vector for chloroplast transformation of a asteraceae plant comprising DNA derived from the chloroplast genome of a asteraceae plant,
(2) The vector according to (1) above, wherein the DNA derived from the asteraceae chloroplast genome is DNA derived from lettuce chloroplasts,
(3) The vector according to (1) or (2), wherein the Asteraceae plant is lettuce,
(4) The vector according to any one of (1) to (3) above, which contains a plurality of DNAs derived from the chloroplast genome of Asteraceae
(5) The vector according to (4) above, wherein the DNA derived from the lettuce chloroplast genome is a ribulose-1,5-biscarboxylase / oxygenase large subunit gene and an acetyl CoA carboxylase subunit gene,
(6) The vector according to any one of (1) to (5) above, which has at least one restriction enzyme cleavage site between DNAs derived from the chloroplast genome of the Asteraceae plant,
(7) having a promoter and terminator that function in the chloroplast between DNA sequences derived from the chloroplast genome of the Asteraceae plant, and further having a base sequence encoding an expressed protein between the promoter and the terminator; The vector according to any one of (1) to (6) above,
(8) The vector according to (7), wherein the promoter is a promoter of rRNA operon derived from a lettuce chloroplast genome,
(9) The promoter of the rRNA operon derived from the lettuce chloroplast genome and the psbA gene between the ribulose-1,5-biscarboxylase / oxygenase large subunit gene and the acetyl CoA carboxylase subunit gene derived from the lettuce chloroplast genome A vector having a terminator and further having a base sequence encoding an expressed protein between the promoter and the terminator,
(10) Asteraceae plant transformed with the vector according to any one of (1) to (9),
(11) Lettuce transformed with the vector according to any one of (1) to (9),
(12) A method for transforming Asteraceae plant chloroplasts, which comprises introducing the vector according to any one of (1) to (9) into a leaf cell of an Asteraceae plant,
(13) The transformation method according to (12), wherein the introduction of the vector uses a particle gun.
(14) The vector according to any one of (1) to (9) is introduced into a leaf cell of an Asteraceae plant, and the leaf cell of the Asteraceae plant is cultured in a plant culture medium. A method for producing a transformed plant,
(15) Auxin 0.01 to 1 mg / L 0.01 wherein the plant culture medium is selected from naphthalene acetic acid, 2-naphthoxyacetic acid, indole acetic acid, 4-chloroindole acetic acid, indole butyric acid and 2,4-dichlorophenoxyacetic acid Containing 1 to 1 mg / L and 0.01 to 1 mg / L of cytokinin selected from kinetin, zeatin or benzyladenine and isopentenyladenine, the method for producing a transformed plant according to (14),
(16) The method for producing a transformed plant according to (15), wherein the weight ratio of cytokinin to auxin is 1: 0.8 to 1.2,
(17) The method for producing a transformed plant according to (15) or (16) above, wherein the plant culture medium further contains 100 to 1000 ppm of polyvinylpyrrolidone,
(18) Auxin 0.01-1 mg / L selected from naphthaleneacetic acid, 2-naphthoxyacetic acid, indoleacetic acid, 4-chloroindoleacetic acid, indolebutyric acid and 2,4-dichlorophenoxyacetic acid, kinetin, zeatin or benzyladenine and A medium for leaf culture of transformed asteraceae plants comprising 0.01-1 mg / L of cytokinin selected from isopentenyl adenine and 100-1000 ppm of polyvinylpyrrolidone;
(19) The medium according to (18) above, wherein the weight ratio of cytokinin to auxin is 1: 0.8 to 1.2, and (20) the asteraceae plant is lettuce, The medium according to (18) or (19),
About.

The vector of the present invention can reliably introduce a gene encoding an expressed protein into the chloroplast of a asteraceae plant.
Among the chloroplasts of asteraceae plants, lettuce chloroplast transformation technology is a chloroplast-transformed plant such as tobacco that has been produced so far due to its low content of harmful secondary metabolites. Compared to the above, it is possible to construct a system capable of producing useful proteins more safely than before. Since lettuce is established by hydroponics, lettuce transformed according to the present invention can be industrially mass-produced using a plant factory, and can produce protein safely and inexpensively. In addition, since it is not affected by weather or pest damage, a stable protein production system can be constructed.
In addition, the asteraceae plant transformed by introducing a metallothionein gene (eg, merA gene) into the chloroplast by the transformation technique of the present invention can be used to detoxify harmful heavy metals or mercury in the environment such as soil. Since it can be detoxified, it can be used for phyto remediation (environmental purification technology using plants). In addition, by introducing the disease damage resistance gene or the herbicide resistance gene into the chloroplast by the transformation technique of the present invention, it is possible to produce a pest resistance or herbicide resistance asteraceae plant. Further, by introducing genes related to biosynthesis such as carotenoids having antioxidative action and vitamin E into the chloroplasts by the transformation technique of the present invention, the sunflowers such as sunflowers and safflowers in the asteraceae plants can be obtained. It is possible to increase the content of useful components such as carotenoids and vitamin E in oils and fats produced by safflower and safflower. Further, by introducing, for example, a fructose 1,6-bisphosphatase gene or sedheptulose 1,7-bisphosphatase gene involved in photosynthesis using the vector of the present invention, a asteraceae plant into which the gene has been introduced is a wild strain. Compared to the above, the height is large, the leaf area is large, it grows quickly, and the ability to synthesize sugar and starch can be increased.
In the Asteraceae plants transformed by the present invention, since the gene encoding the expressed protein is directly introduced into the chloroplast genome instead of the nuclear genome, it is possible to prevent the introduced gene from spreading by pollen. That is, for example, as in a plant in which a gene is introduced into the nucleus, pollen is scattered by a wide range of winds and insects, and environmental pollution such as adverse effects on the animal and plant kingdoms can be prevented.

Examples of the DNA derived from the chloroplast genome of the Asteraceae plant used in the present invention include a ribulose-1,5-biscarboxylase / oxygenase large subunit gene (hereinafter abbreviated as rbcL gene) or an acetyl CoA carboxylase subunit gene. (Hereinafter abbreviated as accD gene).
The rbcL gene is a Rubisco gene encoded in the chloroplast genome. Rubisco 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 turnover in the circuit. The enzyme also catalyzes a reaction for fixing oxygen (O ₂ ) (oxygenase reaction). The rbcL gene is particularly preferably an rbcL gene derived from a lettuce chloroplast genome.
The rbcL gene derived from the lettuce chloroplast genome is determined, for example, from the homology with the already determined base sequence of rbcL of the tobacco chloroplast genome from the whole genome sequence of the lettuce chloroplast. The rbcL gene derived from the lettuce chloroplast genome thus determined and the base sequence in the vicinity thereof are represented by SEQ ID NO: 1 (1640 bp) in the sequence listing. One or more bases in the base sequence of SEQ ID NO: 1, for example, 1 to 150 bases are deleted or replaced with another base, or 1 to 150 other bases Any DNA can be preferably used as long as the DNA modified by adding a base can be homologously recombined with the rbcL gene.

The accD gene is an acetyl CoA carboxylase gene encoded by the chloroplast genome. Acetyl CoA carboxylase is an enzyme involved in fatty acid synthesis in plants. The accD gene is preferably an accD gene derived from a lettuce chloroplast genome.
The accD gene derived from the lettuce chloroplast genome is determined, for example, from the homology with the accD base sequence of the tobacco chloroplast genome which has already been clarified from the entire genome sequence of the lettuce chloroplast. The accD gene derived from the lettuce chloroplast genome thus determined and the base sequence in the vicinity thereof are represented by SEQ ID NO: 2 (1057 bp) in the sequence listing. One or more bases in the base sequence of SEQ ID NO: 2, for example, 1 to 100 bases are deleted or replaced with another base, or 1 to 100 other bases Any DNA can be preferably used as long as the DNA modified by adding a base can homologously recombine with the accD gene.

In the present invention, for example, by using the rbcL gene and the accD gene derived from the lettuce chloroplast genome, the gene encoding the expressed protein introduced into the vector is easily incorporated into the lettuce chloroplast genome by homologous recombination. Furthermore, there is an advantage that the expression level of the expressed protein in the lettuce chloroplast increases.
In the present invention, the rbcL gene and the accD gene derived from the chloroplast genome need not use their full length. For example, the noncoding region between the rbcL gene and the accD gene has a length of about 1000 to 1500 base pairs from the introduction position of the introduced gene to the rbcL gene side or the accD gene side, respectively. Any sequence that can homologously recombine with a gene may be used.

  It is preferable to have at least one, preferably two or more restriction enzyme cleavage sites (hereinafter also referred to as restriction enzyme sites) between the rbcL gene and the accD gene. As the restriction enzyme site, any sequence can be preferably used as long as it includes a site that is recognized and cleaved by the restriction enzyme. Preferred examples of the restriction enzyme site include a PstI site, a NotI site, a SalI site, and an Eco47III site. These restriction enzyme sites can be cleaved with restriction enzymes PstI, NotI, SalI, or Eco47III, respectively.

In addition, between the rbcL gene and the accD gene, a promoter of an rRNA operon (hereinafter abbreviated as Prrn) derived from a chloroplast genome (preferably lettuce) chloroplast genome and a terminator (hereinafter referred to as TpsbA) of a psbA gene. Abbreviated).
For example, Prrn and TpsbA of the Asteraceae plant (preferably lettuce) are derived from homology with Prrn and TpsbA base sequences of tobacco chloroplast genome which has already been clarified from the whole genome sequence of lettuce chloroplasts. It is determined. For example, Prrn derived from the lettuce chloroplast genome thus determined is represented by SEQ ID NO: 3 (113 bp) in the sequence listing, and TpsbA derived from the lettuce chloroplast genome is represented by SEQ ID NO: 4 (339 bp) in the sequence listing. Indicated. In the base sequence of SEQ ID NO: 3 or 4, one or more bases, for example, 1 to 10 bases are deleted, replaced with another base, or 1 to 10 bases Any of the DNAs modified by adding other bases in the above sequence and capable of recognizing the transcription start and transcription termination of the gene encoding the expressed protein can be preferably used.

  The promoter and terminator are not limited to the promoter and terminator as long as they can recognize the transcription start and transcription termination of the gene encoding the expressed protein, and may be other promoters and terminators. Examples of such a promoter include psbA promoter, rbcL promoter, psbD promoter, and atpB promoter. These promoters are also preferably promoters derived from the chloroplast genome, and more preferably promoters derived from the lettuce chloroplast genome. Examples of the terminator include rps16 terminator.

In the present invention, the expression protein is a protein that is desired to be expressed using the expression system according to the present invention, and the type is not particularly limited as long as it is a protein that can be expressed using the expression system. However, it may be a protein derived from a foreign gene. As such expressed proteins, for example, medical proteins having pharmacological activity (eg, insulin, interferon, erythropoietin, interleukin, human somatostatin, tissue plasminogen activator, etc.) or proteins useful as industrial materials are produced. Enzymes (eg, cellulase, nitrile hydratase, etc.) necessary to do so.
In addition, the expressed protein is produced by an enzyme possessed by the plant itself, such as an enzyme involved in photosynthesis (eg, fructose-1,6-bisphosphatase, sedheptulose-1,7-bisphosphatase, transketolase, etc.) or a plant body. Enzymes involved in biosynthesis of useful components (eg carotenoid biosynthetic enzymes), pest-resistant proteins (eg Bacillus thuringes toxin etc.) and herbicide resistant proteins (eg 5-enolpyruvylshikimic acid- And 3-phosphate synthase).
Examples of the expressed protein include metallothionein that binds to harmful heavy metal ions for phytoremediation.

  The vector in the present invention can be constructed by using the pLD6 (SEQ ID NO: 17; 4591 bp) plasmid and pLD200 (SEQ ID NO: 18; 5581 bp) plasmid described in JP-A No. 2002-272476. That is, the Prrn, TpsbA, rbcL gene and accD gene derived from the tobacco chloroplast genome introduced into the pLD6 plasmid and the pLD200 plasmid are converted into Prrn, TpsbA, rbcL gene derived from the asteraceae (preferably lettuce) chloroplast genome and It can be created by replacing with the accD gene. For gene replacement, a Prrn, TpsbA, rbcL gene or accD gene derived from a tobacco chloroplast genome is excised with a restriction enzyme, and instead, a Prrn, TpsbA, rbcL gene derived from an Asteraceae (preferably lettuce) chloroplast genome or This is done by introducing the accD gene.

First, the NotI and Eco47III sites of pLD6 are cleaved using restriction enzymes NotI and Eco47III. Next, the region sandwiched between the restriction enzyme cleavage sites (the region of nucleotides 2220 to 2345 of the sequence described in SEQ ID NO: 17; the region containing Prrn from the tobacco chloroplast genome) is derived from, for example, the lettuce chloroplast genome Replaced with Prrn (SEQ ID NO: 3; 113 bp).
In addition, the PstI and SalI sites of pLD6 were cleaved using restriction enzymes PstI and SalI, and were sandwiched by the restriction enzyme cleavage sites (regions of nucleotides 3174-3913 of the sequence described in SEQ ID NO: 17; tobacco leaf The region containing TpsbA derived from the chloroplast genome) is replaced with, for example, TpsbA derived from the lettuce chloroplast genome (SEQ ID NO: 4; 339 bp). In this manner, a plasmid in which the Prrn and TpsbA sequences derived from the tobacco chloroplast genome of pLD6 are removed and replaced with the base sequences of Prrn and TpsbA derived from the lettuce chloroplast genome can be prepared. The base sequence encoding the expressed protein is arranged between Prrn and TpsbA derived from the lettuce chloroplast genome.

On the other hand, the EcoRI and NotI sites of pLD200 are cleaved by restriction enzymes EcoRI and NotI, and are sandwiched by the restriction enzyme cleavage sites (regions of nucleotides 396 to 2126 of the sequence described in SEQ ID NO: 18; tobacco chloroplasts) The region containing rbcL derived from the genome) is replaced with, for example, rbcL derived from the lettuce chloroplast genome (SEQ ID NO: 1; 1640 bp). Further, the SalI and HindIII sites of pLD200 are cleaved by restriction enzymes SalI and HindIII, and are sandwiched by the restriction enzyme cleavage sites (regions of nucleotides 2141 to 3342 of the sequence described in SEQ ID NO: 18; tobacco chloroplasts) The region containing accD derived from the genome) is replaced with, for example, accD (SEQ ID NO: 2; 1057 bp) derived from the lettuce chloroplast genome. In this manner, a plasmid in which the rbcL and accD sequences derived from the tobacco chloroplast genome of pLD200 are removed and replaced with the base sequences of rbcL and accD derived from the lettuce chloroplast genome can be prepared.

  Next, the base sequence necessary for expression of the expressed protein using restriction enzymes NotI and SalI from pLD6 replaced with the Prrn and TpsbA sequences derived from the lettuce chloroplast genome (nucleotides 2220 to 3913 in the sequence described in SEQ ID NO: 17). Are cut out and introduced into the NotI and SalI sites of pLD200 replaced with the rbcL and accD genes derived from the lettuce chloroplast genome using restriction enzymes NotI and SalI. The region to be introduced is a region corresponding to nucleotides 2127 to 2141 in the sequence described in SEQ ID NO: 18. In this way, it is possible to prepare a vector that causes expression of the expressed protein and homologous recombination in the rbcL and accD gene regions of the lettuce chloroplast genome.

  Further, the vector 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. For example, various drug resistance genes can be mentioned. More specifically, a kanamycin resistance gene (NPT), a spectinomycin resistance gene (aadA), a betaine aldehyde dehydrogenase gene (BADH), etc. are mentioned. Moreover, it is preferable to arrange a promoter and a terminator sequence for controlling the expression of the gene upstream and downstream of the gene, respectively. For example, in order to express the aadA gene in chloroplasts, the aforementioned plant-derived promoters and terminators can be preferably used, but the rrn promoter and the psbA terminator are particularly suitable.

  The vector thus prepared is introduced into a host cell to produce a transformant. At this time, as a host cell, Asteraceae plant cells are preferred, Asteraceae plant leaf cells are more preferred, Asteraceae plant chloroplasts are more preferred, and lettuce chloroplasts are particularly preferred. As a method for transformation by introducing the vector of the present invention into host cells, particularly chloroplasts, known methods such as the particle gun method (Svab, Z., Hajdukiwicz, P., and Maliga, P., Proc. Natl. Acad. Sci. USA, 1990, Vol. 87, p. 8526-8530) or PEG method (Golds, T., Maliga, P., and Koop, H.-U., Bio / Technol., 1993). Year, Vol. 11, p. 95-97) 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 extremely 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.

  Compositae leaf cells into which the vector has been introduced, such as lettuce leaf cells, can be cultured in a plant culture medium to give a plant body. The plant culture medium is obtained by adding a plant hormone to a basic medium containing inorganic salts and vitamins such as Gamburg B5, Murashige-Skoog (MS), Niche & Nitch, and the like. Those are preferred. As the basal medium, MS medium is particularly preferable. As plant hormones, auxin and cytokinin are preferably combined. Examples of auxin include naphthalene acetic acid (hereinafter abbreviated as NAA), 2-naphthoxyacetic acid, indoleacetic acid, 4-chloroindoleacetic acid, indolebutyric acid, or 2,4-dichlorophenoxyacetic acid, and NAA is particularly preferable. Examples of cytokinins include kinetin, zeatin or benzyladenine (hereinafter abbreviated as BA), isopentenyl adenine, and the like, with BA being particularly preferred. The concentration of auxin and cytokinin added to the basal medium varies depending on the auxin and cytokinin used, but the auxin is about 0.01 to 1.0 mg / L, preferably about 0.05 to 0.2 mg / L, more preferably About 0.1 mg / L, cytokinin is added to about 0.01 to 1.0 mg / L, preferably about 0.05 to 0.2 mg / L, and more preferably about 0.1 mg / L. The ratio (weight ratio) of cytokinin to auxin is preferably about 1: 0.8 to 1.2. Further, polyvinyl pyrrolidone (PVP) is added at about 100 to 1000 ppm, preferably about 300 to 700 mg / L, more preferably about 400 to 600 ppm. By adding auxin or cytokinin to the medium, the regeneration of transformed asteraceae plants, especially lettuce, is promoted, and the addition of PVP can prevent the death due to browning during culture.

Moreover, it is preferable to add a saccharide as a carbon source, vitamins, a support for solidifying the medium, and the like to the medium. Examples of the saccharide include sucrose. The amount of saccharide added is about 1 to 10% by weight, preferably about 2 to 5% by weight. Examples of vitamins include thiamine hydrochloride, pyridoxine hydrochloride, nicotinic acid or inositol. Examples of the support include agar, gellan gum, and paper bridge. Although the density | concentration of a support body changes with pH of a culture medium etc., in the case of gellan gum, about 0.2 to 0.3 weight% is preferable, for example.
In addition, an amino acid (eg, glycine), adenine and / or coconut water may be added to the medium.
The medium is usually adjusted to about pH 4-8, preferably about pH 5-7 with potassium hydroxide or the like.

The asteraceae plants transformed by the present invention may be of any kind, for example, lettuce (Lactuca sativa), sunflower (Helianthus annuus), safflower (Carthamus tinctoris), artichoke (Cynara solidmus), burdock (Arc) Chrysanthemum morifolium, Cosmos bipinnatus, Achilla filpendulina, Calendula arbensis, and Daisy (Rudbeckia).
Transformed Asteraceae plants, especially lettuce, can be grown in alleys or hydroponics under conditions known per se. The transformed Asteraceae plant thus prepared, particularly lettuce, can highly express the protein encoded by the gene introduced into the vector. In addition, the transformant of the present invention has advantages such as prevention of scattering to the environment via pollen of the introduced gene.

  The above-described genetic engineering or biotechnological operations are described in commercially available experiments such as Molecular Cloning, Cold Spring Harbor Laboratory, published in 1982, published in 1989. It can be easily carried out according to the method described in Molecular Cloning, 2nd edition (Molecular Cloning, 2nd ed.) Cold Spring Harbor Laboratory.

  Specific examples will be described below in more detail, but the present invention is not particularly limited thereto.

Determination and Analysis of Lettuce Chloroplast Genomic Base Sequence Lettuce chloroplasts are isolated from fresh lettuce leaves by percol density gradient centrifugation according to a conventional method. The genomic DNA of lettuce chloroplasts is purified from the isolated chloroplasts by a DNA purification method using CTAB. A lettuce chloroplast genome library having a DNA fragment derived from chloroplast DNA of about 2 kb is prepared from the purified DNA. The base sequence of 5,428 clones in the genomic library was decoded to determine the lettuce chloroplast whole genome base sequence (152,765 bp). From the homology with the already known tobacco chloroplast genome base sequence, the base sequences of Prbc (arrangement sequence number 2 in the sequence listing) and rbcL (sequence number 1 in the sequence listing) and accD (sequence number 2 in the sequence listing). The nucleotide sequence information of SEQ ID NO: 3) and TpsbA (SEQ ID NO: 4 of the sequence listing) was obtained.

Construction of plasmid vector for transformation of lettuce chloroplast (1) Isolation of lettuce chloroplast genome Prrn, TpsbA Prrn of lettuce chloroplast genome is forward primer:
5′-CCGCGCGCCGCGATTATTTGATTGCTACCC-3 ′ (SEQ ID NO: 5 in the sequence listing),
And reverse primer:
5′-CCAGCGCTATTCGGCCCGGAGTTCGCTCC-3 ′ (SEQ ID NO: 6 in the sequence listing)
Was amplified using.
The Forward primer contains a NotI site at the 5 ′ end and the reverse primer contains an Eco47III site at the 5 ′ end. The amplified fragment was introduced into NotI and Eco47III sites of pLD6 (SEQ ID NO: 17 in the sequence listing) using restriction enzymes NotI and Eco47III. By this operation, the region of nucleotides 2220 to 2345 in the sequence described in SEQ ID NO: 17 of pLD6 was replaced with Prrn (SEQ ID NO: 3 in the sequence listing; 113 bp) derived from the lettuce chloroplast genome.
The terminator of psbA of lettuce chloroplast genome TpsbA is a forward primer:
5′-GGCTGCAGGACTTTGGTCTTATTTGTAAT-3 ′ (SEQ ID NO: 7 in the Sequence Listing)
And reverse primer:
5′-CCGTCGACGAGGCATATTATTTCTTTCTT-3 ′ (SEQ ID NO: 8 in the sequence listing)
Was amplified using.
The Forward primer contains a PstI site at the 5 ′ end, and the reverse primer contains a SalI site at the 5 ′ end. The amplified fragment was introduced into the PstI and SalI sites of pLD6 using restriction enzymes PstI and SalI. By this operation, the region of nucleotides 3174 to 3913 in the sequence described in SEQ ID NO: 17 of pLD6 was replaced with TpsbA derived from the lettuce chloroplast genome (SEQ ID NO: 4 in the sequence listing; 339 bp).
In this way, a pLD6 tobacco-derived promoter and terminator sequence was removed, and a plasmid pRL6 in which the base sequence was derived from a lettuce chloroplast genome was prepared.
As the expressed protein, the spectinomycin resistance gene (hereinafter abbreviated as aadA: SEQ ID NO: 19 in the sequence listing), which was originally introduced into pLD6, was left as it was to obtain the expressed protein in the present invention.

(2) Isolation of rbcL gene and accD gene The 1640 bp region containing the rbcL gene of the lettuce chloroplast genome is a forward primer:
5′-CCGAATTCAATTCATGAGTTGTAGGGAG-3 ′ (SEQ ID NO: 9 in the Sequence Listing)
And reverse primer:
5′-CCGCCGGCCGCGATCCAACCAACACAAAAAT-3 ′ (SEQ ID NO: 10 in the Sequence Listing)
Was amplified using.
The Forward primer contains an EcoRI site at the 5 ′ end, and the reverse primer contains a NotI site at the 5 ′ end. The amplified fragment was introduced into the EcoRI and NotI sites of pLD200 (SEQ ID NO: 18 in the sequence listing) using restriction enzymes EcoRI and NotI. By this operation, the region of nucleotides 396 to 2126 in the sequence described in SEQ ID NO: 18 of pLD200 was replaced with rbcL derived from the lettuce chloroplast genome (SEQ ID NO: 1 in the sequence listing; 1640 bp).
The 1057 bp region containing the accD gene of the lettuce chloroplast genome is the forward primer:
5′-CCGTCGACGATCTCTAGTAGATTGGGATAT-3 ′ (SEQ ID NO: 11 in the sequence listing) and reverse primer:
Amplification was performed using 5′-GGAAGCTTCCCATATGAGTAGACTACTTC-3 ′ (SEQ ID NO: 12 in the Sequence Listing).
The Forward primer contains a SalI site at the 5 ′ end, and the reverse primer contains a HindIII site at the 5 ′ end. The amplified fragment was introduced into the SalI and HindIII sites of pLD200 using restriction enzymes SalI and HindIII. By this operation, the region of nucleotides 2141 to 3342 of the sequence described in SEQ ID NO: 18 of pLD200 was replaced with accD derived from the lettuce chloroplast genome (SEQ ID NO: 2; 1057 bp in the sequence listing).
Thus, the pLD200 tobacco-derived rbcL and accD-encoding sequences were removed, and a plasmid pRL200 was prepared in which the nucleotide sequences encoding the lettuce chloroplast genome-derived rbcL and accD were replaced.

(3) Preparation of lettuce chloroplast transformation vector DNA fragment necessary for aadA gene expression from restriction plasmid NotI and SalI from the plasmid (1) introduced with a promoter and terminator sequence derived from the lettuce chloroplast genome (A region corresponding to nucleotides 2220 to 3913 of the sequence described in SEQ ID NO: 17) was excised. The excised DNA fragment was introduced into the NotI and SalI sites of the plasmid (2) into which the rbcL and accD genes derived from the lettuce chloroplast genome were introduced using restriction enzymes NotI and SalI. The region into which the DNA fragment is introduced is a region corresponding to nucleotides 2127 to 2141 of the sequence set forth in SEQ ID NO: 18.
In this way, a lettuce chloroplast transformation vector pRL1000 was produced that caused the expression of the aadA gene and homologous recombination in the rbcL and accD gene regions of the lettuce chloroplast genome (FIG. 1).

Transformation and redifferentiation of lettuce chloroplasts Live seed leaves of aseptically cultivated lettuce for about 3 to 4 weeks after seeding in MS medium are used for gene transfer. MS medium is MS salt, inositol (100 mg / L), thiamine hydrochloride (0.1 mg / L), pyridoxine hydrochloride (0.5 mg / L), nicotinic acid (0.5 mg / L), glycine (2 mg / L) , Sucrose (30 g / L) and gellan gum (2 g / L), adjusted to pH 5.8 with KOH. Five plates are prepared by placing 6 to 7 fresh leaves of lettuce on RMOP medium and pre-cultured on the medium for 1 day. The composition of the RMOP medium is obtained by adding BA (0.1 mg / L) and NAA (0.1 mg / L) to the MS medium. The mixture is mixed with 2.3 mg of gold particles having a diameter of 0.6 micron, 25 μg of lettuce chloroplast transformation vector, 2.5 M calcium chloride and 0.1 M spermidine and suspended at 4 ° C. for 10 minutes every minute. After washing twice with ethanol, it is suspended in 60 μL of ethanol. This corresponds to an amount that can be used for 10 driving experiments. Gold particles are implanted into the lettuce leaf cells using a particle gun (model PDS-1000 / He BIO-RAD) on the pre-cultured lettuce leaves. Five shots are performed per experiment. The driving pressure is 900 psi. Two days after the implantation, the fresh leaves into which the vector has been implanted are cut into 4 mm square sections with a scalpel and placed on an RMOP medium containing spectinomycin (50 mg / L) and polyvinylpyrrolidone (500 mg / L). A green callus was generated from a leaf section whitened in about 3 to 4 weeks, and then redifferentiated to regenerate a plant individual.

Analysis of Lettuce Chloroplast Transformant by PCR About 50 mg of the leaf tissue of the lettuce plant regenerated in Example 3 is collected and crushed under liquid nitrogen. A DNA extraction buffer (0.3 M sodium chloride, 0.05 M Tris-hydrochloric acid, pH 7.5, 20 mM EDTA, 0.5% SDS, 5 M urea, 5% phenol) was added to the crushed sample and mixed. Extraction with phenol / chloroform [1/1 (V / V)], precipitation with ethanol, and air drying. The extracted DNA was dissolved in 100 μL of TE solution [10 mM Tris-HCl (pH 8.0), 1 mM EDTA].
In PCR for confirming the presence or absence of the aadA sequence, a forward primer with a DNA solution extracted from a lettuce plant as a template:
5′-ATGGCTCGGTGAAGCGGTAT-3 ′ (SEQ ID NO: 13 in the Sequence Listing)
And reverse primer:
5′-TTATTTGCCCAACTACTCTTAG-3 ′ (SEQ ID NO: 14 in the Sequence Listing)
A PCR reaction was carried out using and confirmed by agarose gel electrophoresis. As controls, the pRL1000 vector prepared in Example 2 and the wild lettuce leaf tissue were treated in the same manner. The results are shown in FIG. A 0.8 kb band indicating the presence of the aadA gene was observed in the pRL1000 vector and the transformed lettuce.
In addition, the DNA solution extracted from each sample can be used to examine the abundance of the chloroplast genome introduced in the transformed lettuce tissue and the chloroplast genome remaining in the wild type without gene introduction. Forward template:
5′-AGGATTGAGCCCGAATCCAAC-3 ′ (SEQ ID NO: 15 in the Sequence Listing)
And reverse primer:
5′-AGGATTTGTTCTCTCCTACG-3 ′ (SEQ ID NO: 16 in the Sequence Listing)
PCR was performed using As a control, the pRL200 vector prepared in Example 2 and the genomic DNA extracted from the leaf tissue of wild lettuce were treated in the same manner. The results are shown in FIG. A band indicating the introduction of a gene into the 1.6 kb chloroplast genome was confirmed in the lane obtained by electrophoresis of the PCR product using the pRL1000 vector (lane A) and genomic DNA extracted from the transformation lettuce (lane C) as a template. did it. In addition, although a slight amount of a 0.3 kb band indicating the presence of the wild-type chloroplast genome was observed in lane C, a trace amount of wild-type chloroplast that can be detected by PCR in the transformed lettuce. It was confirmed that genomic DNA was also included (FIG. 3).

Evaluation of spectinomycin resistance of transformed lettuce Lettuce transformed to examine whether the aadA gene is expressed and functioning in a lettuce plant in which introduction of the aadA gene has been confirmed according to Example 4 (Fig. From 4), 4 mm square leaf sections were cut out and placed on RMOP medium containing 50 mg / L of spectinomycin. Similarly, leaves of wild-type lettuce were placed on the floor, and the state of redifferentiation was observed for 4 weeks under conditions of 25 ° C. and long days (16 hours light period, 8 hours dark period) (FIG. 5). A, B, and C are obtained by culturing leaf sections of wild-type lettuce on an RMOP medium containing spectinomycin. Although A shows the state of the leaf section immediately after placement, B shows the state of the leaf after 2 weeks, and C shows the state of the leaf section after the placement of 4 weeks, no differentiation of leaves and roots was observed. D, E, and F show the culture of leaf sections of the transformed lettuce in the same manner. In E and F, leaf and root differentiation from leaf sections was confirmed. This shows that the aadA gene was introduced into the chloroplast genome of lettuce, and the aadA product expressed in the leaves showed resistance to spectinomycin.

  High-value-added proteins for medical use can be produced safely and inexpensively from asteraceae plants, especially lettuce chloroplasts.

FIG. 1 is a schematic diagram of a vector. FIG. 2 is a diagram showing the results of agarose gel electrophoresis of the PCR product confirming that the aadA gene was introduced in Example 4. In the figure, A is the DNA extracted from the pRL1000 vector, B is the DNA extracted from the leaves of wild lettuce, and C is the DNA extracted from the transformed lettuce leaves as a template. The results are shown. FIG. 3 is a diagram showing the results of agarose gel electrophoresis of PCR products in which the recombination rate of the transgene into the lettuce chloroplast genome in Example 4 was examined. In the figure, A shows DNA extracted from the pRL1000 vector, B shows DNA extracted from wild lettuce leaves, and C shows DNA extracted from transformed lettuce leaves. The result of electrophoresis is shown. FIG. 4 is a transformed lettuce leaf. FIG. 5 is a diagram showing the results of Example 5.

Claims (7)

And ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit gene having the nucleotide sequence of SEQ ID NO: 1, between the acetyl CoA carboxylase subunit gene having the nucleotide sequence of SEQ ID NO: 2, the nucleotide sequence of SEQ ID NO: 3 A vector for transforming lettuce chloroplasts, which has a promoter of the rRNA operon consisting of the terminator and a terminator of the psbA gene consisting of the base sequence of SEQ ID NO: 4 , and further has a base sequence encoding an expressed protein between the promoter and the terminator. A method for transforming lettuce chloroplasts, which comprises introducing the vector according to claim 1 into lettuce leaf cells. The transformation method according to claim 2 , wherein the introduction of the vector is performed using a particle gun. A method for producing transformed lettuce , wherein the vector according to claim 1 is introduced into lettuce leaf cells, and the lettuce leaf cells are cultured in a plant culture medium. Media for plant culture, naphthalene acetic acid, 2-naphthoxy acetic acid, indole acetic acid, 4-chloro indole acid, auxin 0.01 to 1 mg / L selected from indole butyric acid and 2,4-dichlorophenoxyacetic acid, kinetin in parallel beauty, The method for producing transformed lettuce according to claim 4 , comprising 0.01-1 mg / L of cytokinin selected from zeatin or benzyladenine and isopentenyladenine. The method for producing transformed lettuce according to claim 5 , wherein the weight ratio of cytokinin to auxin is 1: 0.8 to 1.2. The method for producing transformed lettuce according to claim 5 or 6 , wherein the plant culture medium further contains 100 to 1000 ppm of polyvinylpyrrolidone.