WO2006027865A2

WO2006027865A2 - Method of transforming chloroplast of composite plant

Info

Publication number: WO2006027865A2
Application number: PCT/JP2005/004899
Authority: WO
Inventors: Ken-Ichi Tomizawa; Hirosuke Kanamoto; Akiho Yokota; Hiroshi Asao
Original assignee: Research Institute Of Innovative Technology For The Earth; National University Corporation NARA Institute of Science and Technology
Priority date: 2004-09-09
Filing date: 2005-03-14
Publication date: 2006-03-16
Also published as: WO2006027865A3; JP2006075078A; JP4740567B2

Abstract

An object of present invention is to provide a transformation method which can effectively express a useful protein such as a medical protein in a composite plant chloroplast without producing a harmful secondary metabolite. A vector having a promoter of RNA operon derived from a composite plant chloroplast genome and a terminator of a psbA gene between a ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit gene and an acetyl CoA carboxylase subunit gene, and having a nucleotide sequence encoding an expression protein between the promoter and the terminator is introduced into a chloroplast of a composite plant.

Description

DESCRIPTION

METHOD OF TRANSFORMING CHLOROPLAST OF COMPOSITE PLANT

Technical Field

The present invention relates to a method of transforming a composite plant chloroplast.

Background Art Currently, regarding a process for industrially producing a protein, studies on the preparation of medical proteins by introducing a gene expressing a protein useful as a medicine into a nuclear chromosome of Escherichia coli or a plant and expressing the gene have already progressed in several countries. However, when a bacterium such as Escherichia coli is used, toxic contamination into final products can not be avoided. In addition, a process for producing a protein by a transformed plant in which a gene has been transferred into a plant nuclear genome has been developed, but there remains a problem such as low amount of the protein production. Further, in the case of transferring a gene into a plant nucleus, gene dispersion into the natural world by pollens flying is regarded as a problem. Under such situation, a method of transformation into a plant chloroplast genome has been put into practice by a tobacco by Maliga et al. Chloroplast transformation has a great characteristic that a chloroplast has ability to produce and accumulate a large number of proteins as compared with previouslyperformed transformation into aplant nuclear genome. Examples of other characteristics in the chloroplast transformation include the fact that genetic information of a chloroplast is generally maternally inherited, and a possibility of horizontal dispersion of the introduced genes via apollen is low, and an advantage of multiple gene expression utilizing polycistronic control of a chloroplast. In view of these advantages, increasing attention is being paid to a transformed chloroplast as a useful system for producing a substance such as a protein. For example, a vector which can highly express a target protein in a tobacco chloroplast is known. This vector is characterized in that it has a psbA promoter, and a ribosome binding site upstream of a translation initiation point of a gene encoding a protein (see JP-A-2002-272476) . This method is aimed at preparing a protein having pharmacological activity or a protein useful as a material for medicine industry, employing tobacco in place of the preparation by microorganisms. However, since in this method, a plant to be transformed is tobacco, a substance harmful to the body is also produced as a secondary metabolite of tobacco and, therefore, it can be said that this method is not suitable for producing a protein requiring high purification such as a medical protein.

With respect to transformation of a composite plant, there have been known, for example, lettuce big-vein virus resistant lettuce obtained by introducing a DNA suppressing production and function of a lettuce big-vein virus protein into a lettuce cell (see JP-A-2002-272476), and lettuce transformed using a plant transformation vector containing a gene of a lettuce-infective yellow virus protein or a part of the gene (International Publication WO 01/090362). However, in the case of any one of these transformed lettuces, each gene is introduced into a nuclear genome of the leaf by introducing a plasmid constructed using the gene into Agrobacterium, and infecting the leaf disc with the microorganism, and thus the gene is not introduced directly into the chloroplast.

Disclosure of the Invention An object of the present invention is to provide a method of transformation which can effectively express a useful protein such as medical proteins in a composite plant chloroplast without producing a harmful secondary metabolite.

The present inventors paid an attention to a lettuce (Laσtuca sativa) which is a composite plant, as a plant having a low possibility to produce a harmful secondary metabolite. In addition, since lettuce can be also cultivated in a plant factory utilizing hydroponics, the present inventors tried to construct a stable system for producing a protein by use of lettuce which is not influenced on weather or damage by disease or insect pests. For this reason, the present inventors intensively studied a process for producing a protein in a lettuce chloroplast. First, in order to obtain a nucleotide sequence derived from a lettuce chloroplast and study aposition into which a foreign gene is introduced, the present inventors first determined a whole nucleotide sequence of the lettuce chloroplast. From the determined nucleotide sequence of the lettuce chloroplast, the present inventors constructed avector having a homologous sequence (ribulose-1,5- bisphosphate carboxylase/oxygenase large subunit gene and acetyl CoA carboxylase subunit gene) which allows for effectivehomologous recombination with a lettuce chloroplast genome, and a lettuce chloroplast genome-derived expression regulating sequence (promoter of rRNA operon and terminator of psbA gene) for assuredly expressing an introduced gene in a lettuce chloroplast. Further, the present inventors continued to study amethod of transferring a gene into a lettuce chloroplast genome utilizing a constructed vector. In addition, the present inventors have noticed the importance of the step where a leaf cell of lettuce in which a gene is introduced into a lettuce chloroplast genome reproduces into a plant individual, and studied the conditions for redifferentiation of a strip of lettuce leaf in which a gene is introduced into a lettuce chloroplast genome. They further studied a method of cultivating the thus obtained plant individual and finally completed the present invention.

That is, the present invention relates to: (1) a vector for transforming a composite plant chloroplast, comprising a DNA derived from a composite plant chloroplast genome,

(2) the vector according to the above (1), wherein the DNA derived from a composite plant chloroplast genome is a DNA derived from a lettuce chloroplast,

(3) the vector according to the above (1) or (2) , wherein the composite plant is a lettuce,

(4) the vector according to any one of the above (1) to (3) , which contains a plurality of DNAs derived from a composite plant chloroplast genome,

(5) the vector according to the above(4), wherein the DNA derived from a lettuce chloroplast genome is a ribulose-1,5- bisphosphate carboxylase/oxygenase large subunit gene and an acetyl CoA carboxylase subunit gene,

(6) the vector according to any one of the above (1) to (5) , which comprises at least one kind of a restriction enzyme cleavage site between DNAs derived from a composite plant chloroplast genome,

(7) the vector according to any one of the above (1) to (6), wherein a promoter and a terminator functioning in a chloroplast between DNA sequences derived from a composite plant chloroplast genome, and has a nucleotide sequence encoding an expression protein between the promoter and the terminator,

(8) the vector according to the above (7) , wherein the promoter is a promoter of rRNA operon derived from a lettuce chloroplast genome, (9) a vector having a promoter of rRNA operon derived from a lettuce chloroplast genome and a terminator of a psbA gene between a ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit gene and an acetyl CoA carboxylase subunit gene derived from a lettuce chloroplast genome, and having a nucleotide sequence encoding an expression protein between the promoter and the terminator,

(10) a composite plant, which is transformed using a vector as defined in any one of the above (1) to (9) , (11) a lettuce, which is transformed using a vector as defined in any one of the above (1) to (9) ,

(12) amethod of transforming a composite plant chloroplast, which comprises introducing a vector as defined in any one of the above (1) to (9) into a leaf cell of a composite plant,

(13) the transformationmethod according to the above (12) , wherein a vector is introduced using a particle gun,

(14) a process for producing a transformed plant, which comprises introducing avector as defined in anyone of the above (1) to (9) into a leaf cell of a composite plant, and culturing the leaf cell of a composite plant in a plant culture medium,

(15) the process for producing a transformed plant according to the above (14), wherein the plant culture medium contains 0.01 to 1 mg/L of an auxin selected from naphthaleneacetic acid, 2-naphthoxyacetic acid, indoleacetic acid, 4-chloroindoleacetic acid, indolebutyric acid and 2,4-dichlorophenoxyacetic acid, and 0.01 to 1 mg/L of a cytokinin selected from kinetin, zeatin, benzyladenine and isopentenyladenine, (16) the process for producing a transformed plant according to the above (15), wherein the ratio by weight of cytokinin relative to auxin is 1 : 0.8 to 1.2,

(17) the process for producing a transformed plant according to the above (15) or (16), wherein the plant culture medium contains 100 to 1000 ppm of polyvinylpyrrolidone,

(18) a medium for culturing a leaf of a transformed composite plant comprising 0.01 to 1 mg/L of an auxin selected from naphthaleneacetic acid, 2-naphthoxyacetiσ acid. indoleacetic acid, 4-chloroindoleacetic acid, indolebutyric acid and 2,4-dichlorophenoxyacetic acid, 0.01 to 1 mg/L of a cytokinin selected from kinetin, zeatin, benzyladenine and isopentenyladenine, and 100 to 1000 ppm of polyvinylpyrrolidone,

(19) the medium according to the above (18), wherein the ratio by weight of cytokinin relative to auxin is 1 : 0.8 to 1.2, and

(20) the medium according to the above (18) or (19) , wherein the composite plant is a lettuce.

The vector of the present invention can assuredly transfer a gene encoding an expression protein into a chloroplast of a composite plant.

Since in the technique of transforming a chloroplast of composite plants, particularly a lettuce chloroplast, the content of harmful secondary metabolites is low, it becomes possible to construct a system which can produce a useful protein more safely than usual as compared with a chloroplast-transformed plant such as tobacco which has been previously produced. In addition, since a method of hydroponics has been established in lettuces, a lettuce transformed by the present invention can be industrially produced at a large scale utilizing a plant factory, therefore, a protein can be produced safely and at a low cost. Further, since its production is not influenced by weather and damage by disease or insect pests, a system for safely producing a protein can be constructed.

In addition, since by technique of transformation of the present invention, a composite plant transformedby introducing a metallothionein gene (e.g. merA gene, etc.) into the chloroplast can make harmless or detoxify harmful heavy metals or mercury in environment such as soil, it can be utilized in phytoremediation (technique of cleaning environment utilizing a plant) . In addition, by introducing a disease-resistant gene, a pest-resistant gene, an insect-resistant gene or a herbicide-resistant gene into a chloroplast according to the transformation technique of the present invention, a disease-resistant, pest-resistant, insect-resistant or herbicide resistant composite plant can be produced. Further, by introducing a gene associated with biosynthesis of carotenoid or vitamin E having antioxidant action into a chloroplast by the transformation technique of the present invention, it becomes possible to increase the content of a useful component such as carotenoid or vitamin E in fats or oils produced by sunflower or safflower in a composite plant such as sunflower or safflower. In addition, by introducing a fructose 1,6-bisphosphatase gene or a sedoheptulose 1,7- bisphosphatase gene associated with photosynthesis using the vector of the present invention, a composite plant with the gene introduced therein is taller, has larger leaves, grows faster, and has increased ability to synthesize sugars or starch as compared with a wild strain. In a composite plant transformed by the present invention, since a gene encoding an expression protein is not in a nuclear genome, but is directly introduced into a chloroplast genome, it is possible to prevent dispersion via pollens of introduced genes. That is, an environmental pollution such as adverse influence on the animal and plant world caused by wide scattering of pollens by wind or an insect, like in the case of a plant in which a gene is introduced into a nucleus, can be prevented.

Brief Description of the Drawings

Fig. 1 is a view schematically showing a vector;

Fig. 2 is a view showing results of agarose gel electrophoresis of a PCR product for confirming that an aadA gene in Example 4 has been introduced. A in the figure denotes results of electrophoresis of a product from PCR using, as a template, a DNA extracted from a pRLIOOO vector, B denotes results usingaDNAextractedfromaleaf ofwild strain lettuce, and C denotes results using a DNA extracted from a leaf of transformed lettuce;

Fig. 3 is a view showing results of agarose gel electrophoresis of a PCR product on which a recombination rate of a gene introduced into a lettuce chloroplast genome in Example 4 was studied. In this figure, A denotes results of electrophoresis of a product from PCR using, as a template, a

DNA extracted from a pRLIOOO vector, B denotes results using aDNA extracted froma leaf ofwild strain lettuce, and C denotes results using a DNA extracted from a leaf of transformed lettuce;

Fig. 4 is a leaf of transformed lettuce; and

Fig. 5 is a view showing results of Example 5. Best Mode for Carrying Out the Invention

Examples of aDNAderived froma composite plant chloroplast genome used in the present invention include a ribulose-l,5-bisphosphate carboxylase/oxygenase large subunit gene (hereinafter abbreviated as rbcL gene) and an acetyl CoA carboxylase subunit gene (hereinafter abbreviated as aσcD gene) .

The rbcL gene is a gene of Rubisco encoded by a chloroplast genome. Rubisco is a key enzyme which catalyses a CO2 fixation reaction (carboxylase reaction) which is an initial stage, in a photosynthesis CO₂ fixation reaction cycle (Calvin cycle) , and is rate-limiting in metabolism turnover in the cycle. In addition, the enzyme also catalyses an oxygen (O₂) fixation reaction (oxygenase reaction). As the rbcL gene, inter alia, a rbcL gene derived from a lettuce chloroplast genome is preferable.

The rbcL gene derived from a lettuce chloroplast genome is determined from homology with a nucleotide sequence of rbcL of a tobacco chloroplast genome which has already been elucidated, for example, from a lettuce chloroplast whole genome nucleotide sequence. The thus determined rbcL gene derived from a lettuce chloroplast genome and its adjacent nucleotide sequence are shown by SEQ ID NO: 1 (1640 bp) of Sequence Listing. If a DNA alternated by deleting one or a plurality of bases, for example, 1 to 150 bases in the nucleotide sequence in SEQ ID NO: 1, or substituting those bases with other bases, or adding other 1 to 150 bases is a sequence which can be homologously recombinated with a rbcL gene, such DNA can be preferably used.

The accD gene is a gene of acetyl CoA carboxylase encoded by a chloroplast genome. The acetyl CoA carboxylase is an enzyme involved in the synthesis of fatty acids in a plant. As the accD gene, an accD gene derived from a lettuce chloroplast genome is preferable.

The accD gene derived from a lettuce chloroplast genome can be determined from homology with a nucleotide sequence of accD of a tobacco chloroplast genome which has already been elucidated, for example, from a lettuce chloroplast whole genome nucleotide sequence. The thus determined accD gene derived from a lettuce chloroplast genome and its adjacent nucleotide sequence are shown by SEQ ID NO: 2 (1057 bp) of Sequence Listing. If a DNA altered by deleting one or a plurality of bases, for example, 1 to 100 bases in a the nucleotide sequence of SEQ ID NO: 2, or substituting those bases with other bases, or adding other 1 to 100 bases is a sequence which can be homologously recombinated with an accD gene, such DNA can be preferably used. In the present invention, by using, for example, a rbcL gene and an accD gene derived from a lettuce chloroplast genome, there is an advantage that a gene encoding an expression protein to be transferred into a vector becomes easy to be incorporated into a lettuce chloroplast genome by homologous recombination, and an expression level of an expression protein in a lettuce chloroplast is increased.

In addition, in the present invention, it is not necessary to use a full length of a rbcL gene and an accD gene derived from a chloroplast genome. For example, a sequence which has a length of each about 1000 to 1500 base pairs on a rbcL gene side or an accD gene side from the position of introduction of a gene to be introduced, of a non-coding region between a rbcL gene and an accD gene, and which can be homologously recombinated with a rbcL gene or an accD gene may be used.

It is preferable that at least one kind, preferably two or more kinds of restriction enzyme cleavage sites (hereinafter, also referred to as restriction enzyme site) are possessed between the rbcL gene and the accD gene. Any sequence of a restriction enzyme site can be preferably used as far as it is a sequence containing a site which is recognized and cut by a restriction enzyme. Preferable examples of the restriction enzyme site include a Pstl site, a Notl site, a Sail site, and an Eco47III site. These restriction enzyme sites can be cut with restriction enzymes Pstl, Notl, Sail and Eco47III, respectively.

In addition, a promoter of rRNA operon (hereinafter, abbreviated as Prrn) derived from a composite plant (preferably lettuce) chloroplast genome and a terminator of a psbA gene (hereinafter, abbreviated as TpsbA) are possessed between the rbcL gene and the accD gene.

The aforementioned Prrn and TpsbA of a composite plant (preferably lettuce) are determined from the homology with a nucleotide sequence of Prrn and TpsbA of a tobacco chloroplast genomewhichhas alreadybeen elucidated. For example, the thus determined Prrn derived from a lettuce chloroplast genome is shown by SEQ ID NO: 3 (113 bp) of Sequence Listing, and TpsbA derived from a lettuce chloroplast genome is shown by SEQ ID NO: 4 (339 bp) of Sequence Listing. Any sequence can be preferably used as far as it is a sequence of a DNA altered by deleting one or a plurality of bases, for example, 1 to 10 bases among a nucleotide sequence of SEQ ID NO: 3 or 4, substituting those bases with other bases, or adding other 1 to 10 bases, and in which transcription initiation and transcription termination of a gene encoding an expression protein are recognized, respectively. A promoter and a terminator are not limited to the aforementioned promoter and terminator as far as they can recognize transcription initiation and transcription termination of a gene encoding an expression protein, and other promoter and terminator may be used. Examples of such promoter include a psbA promoter, a rbcL promoter, a psbD promoter and an atpB promoter. Among these promoters, a promoter derived from a chloroplast genome is preferable, and a promoter derived from a lettuce chloroplast genome is more preferable. Examples of the terminator include a rpslβ terminator.

In the present invention, an expressionprotein is aprotein which is intended to be expressed using an expression system related to the present invention. A kind thereof is not particularly limited as far as it is a protein which can be expressed using the present expression system, and a protein inherent to a composite plant, and a protein derived from a foreign gene may be included. Examples of such expression protein include a medical protein having pharmacological activity (e.g. insulin, interferon, erythropoietin, interleukin, human somatostatin, tissue plasminogen activator etc.) and an enzymes (e.g. cellulase, nitrilehydratase etc.) necessary for preparing a protein useful as an industrial material.

Examples of the expression protein include an enzyme possessed by a plant itself, for example, an enzyme involved in photosynthesis (e.g. fructose-1,6-bisphosphatase, sedoheptulose-l,7-bisphosphatase, transketolase etc. ) and an enzyme involved in biosynthesis of a useful component produced in a plant (e.g. carotenoid biosynthesis enzyme etc.), and a disease-resistant or insect pest-resistant protein (e.g. Bacillus thuringiensis toxin etc.) and a herbicide resistant protein (e.g. 5-enolpyruvylshikimic acid-3-phosphate synthesis enzyme etc.).

In addition, examples of the expression protein include metallothionein which is bound to a harmful heavy metal ion for phytoremediation.

A vector in the present invention can be constructed utilizing a pLD6 (SEQ ID NO: 17; 4591 bp) plasmid and a pLD200 (SEQ ID NO: 18; 5581 bp) plasmid described in JP-A-2002-272476. That is, the vector can be made by substituting Prrn, TpsbA, and rbcL and accD genes derived from a tobacco chloroplast genome, which are introduced into a pLD6 plasmid and a pLD200 plasmid, with Prrn, TpsbA, rbcL, and accD genes derived from a composite plant (preferably lettuce) chloroplast genome. Substitution of a gene is performed by excising a Prrn, TpsbA, rbcL, or accD gene derived from a tobacco chloroplast genome with a restriction enzyme and, instead, introducing a Prrn, TpsbA, rbcL, or accD gene derived from a composite plant (preferably lettuce) chloroplast genome.

First, Notl and Eco47III sites of pLD6 are cut using restriction enzymes Notl andEco47III. Then, aregion (aregion of nucleotides 2220 to 2345 of a sequence described in SEQ ID NO: 17; a region containing a Prrn gene derived from a tobacco chloroplast genome) held by the restriction enzyme cleavage sites is substituted, for example, with a Prrn gene (SEQ ID NO: 3; 113 bp) derived from a lettuce chloroplast genome.

In addition, a Pstl and Sail site of pLD6 is cut using restriction enzymes Pstl and Sail, and a region (region of nucleotides 3174 to 3913 of a sequence described in SEQ ID NO: 17; region containing TpsbA derived from tobacco chloroplast genome) held by the restriction enzyme cleavage sites is substituted, for example, with TpsbA (SEQ ID NO: 4; 339 bp) derived from a lettuce chloroplast genome. Like this, Prrn and TpsbA sequences derived from a tobacco chloroplast genome of pLD6 can be removed to make a plasmid in which those sequences are substituted with nucleotide sequences of Prrn and TpsbA derived from a lettuce chloroplast genome. A nucleotide sequence encoding an expression protein is disposed between Prrn and TpsbA derived from a lettuce chloroplast genome.

On the other hand, an EcoRI and Notl site of pLD200 is cut with restriction enzymes EcoRI and Notl, and a region (region of nucleotides 396 to 2126 of sequence described in SEQ ID NO: 18; region containing rbcL derived from tobacco chloroplast genome) held by the restriction enzyme cleavage sites is substituted, for example, with rbcL (SEQ ID NO: 1; 1640 bp) derived from a lettuce chloroplast genome. In addition, a Sail and HindIII site of pLD200 is cut with restriction enzymes Sail and HindIII, and a region (a region of nucleotides 2141 to 3342 of a sequence described in SEQ ID NO: 18; region containing accD derived from a tobacco chloroplast genome) is substituted, for example, accD (SEQ ID NO: 2; 1057 bp) derived from a lettuce chloroplast genome. Like this, rbcL and accD sequences derived from a tobacco chloroplast genome of pLD200 can be removed to make a plasmid in which those sequences are substituted with nucleotide sequences of Prrn and TpsbA derived from a lettuce chloroplast genome.

Then, a nucleotide sequence necessary for expression of an expression protein (a region corresponding to nucleotides 2220 to 3913 in a sequence described in SEQ ID NO: 17) is excised from pLD6 inwhich sequences are substitutedwith Prrn and TpsbA sequences derived form a lettuce chloroplast genome using restriction enzymes Notl and Sail, and is introduced into a Notl and Sail site of pLD200 in which genes are substituted with rbcL and accD genes derived from a lettuce chloroplast genome using restriction enzymes Notl and Sail. The region to be introduced is a region corresponding to nucleotides 2127 to 2141 in a sequence described in SEQ ID NO: 18. Like this, a vector which generates protein expression and homologous recombination at a region of rbcL and accD genes of a lettuce chloroplast genome can be made.

In addition, it is preferable that the vector has a gene for screening transformants. The gene for screening transforraants is not particularly limited, but the genes known per se may be used. Examples of such genes include various drug resistant genes. More specifically, such resistant genes include a kanamycin resistant gene (NPT) , a spectinomycin resistant gene (aadA) , and a betaine aldehyde dehydrogenase gene (BADH) . It is preferable that a promoter sequence and a terminator sequence for controlling expression of the gene are disposedupstream or downstream of the gene, respectively. For example, in order to express an aadA gene in a chloroplast, the plant-derived promoter and terminator can be preferably used, and a rrn promoter and a psbA terminator are particularly preferable.

The thus made vector is introduced to prepare a transformant. Thereupon, as a host cell, a composite plant cell is preferable, a composite plant leaf cell is more preferable, a chloroplast of a composite plant is further preferable, and a lettuce chloroplast is particularly preferable. As a method of transformation by introducing the vector of the present invention into a host cell, particularly a chloroplast, the known method, for example, a particle gun method (Svab, Z., Hajdukiewicz, P., and Maliga, P., Proc. Natl. Acad. Sci. USA, 1990, vol.87, p.8526-8530) and a PEGmethod (Golds. , T. , Maliga, P., and Koop, H. -U. ,Bio/Technol. , 1993, 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 covering an extremely fine particle of gold or tungsten with a vector, and shooting a particle with the vector attached thereto into ahost cell by a gunpowder or a high pressure gas. A leaf cell of a composite plant in which a vector has been introduced, for example, a lettuce leaf cell can be cultured into a plant in a plant culture medium. A medium for culturing a plant is preferably such that a plant hormone is added to a basal medium containing an inorganic salt and vitamins, such as Gamborg B5, Murashige-Skoog, MS, and Nitch&Nitch. As the basal medium, in particular, a MS medium is preferable. As a plant hormone, it is better to combine an auxin and a cytokinin. Examples of the auxin include naphthaleneacetic acid (hereinafter abbreviated as NAA), 2-naphthoxyacetic acid, indoleacetic acid, 4-chloroindoleacetic acid, indolebutyric acid and 2,4-dichlorophenoxyacetic acid, and NAA is particularly preferable. Examples of the cytokinin include kinetin, zeatin or benzyladenine (hereinafter abbreviated as BA) , and isopentenyladenine, andBA is particularlypreferable. Although concentration of the auxin and the cytokinin to be added to a basal medium is different depending on auxins and cytokinins to be used, auxin is added so that a concentration becomes about 0.01 to 1.0 mg/L, preferably about 0.05 to 0.2 mg/L, further preferably about 0.1 mg/L, and cytokinin is added so that a concentration becomes to be about 0.01 to 1.0 mg/L, preferably about 0.05 to 0.2 mg/L, more preferably about 0.1 mg/L. The ratio (ratio by weight) of a cytokinin relative to an auxin is preferably about 1:0.8 to 1.2. It is better to add about 100 to 1000 ppm, preferably about 300 to 700 mg/L, more preferably about 400 to 600 ppm of polyvinylpyrrolidone (PVP) to a medium. By adding an auxin and a cytokinin to a medium, redifferentiation of a transformed composite plant. particularly lettuce, is promoted, and withering due to browning at culturing can be prevented by addition of PVP.

In addition, it is preferable to add saccharides as a carbon source, vitamins, and a solidifying reagent to the aforementioned medium. Examples of saccharides include sucrose. An amount of saccharides to be 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 and inositol. Examples of the solidifying reagent include agar, gellan gum and paper bridge. Although concentration of a solidifying reagent is different depending on the pH of medium, in the case of gellan gum, for example, about 0.2 to 0.3% by weight is preferable.

In addition, amino acid (e.g. glycine, etc.), adenine and/or coconut water may be added to the medium.

The medium is adjusted to a pH of about 4 to 8, preferably a pH of about 5 to 7 usually with potassium hydroxide and the like.

A composite plant to be transformedby the present invention may be any kind, and examples of such plants include lettuce

(Lactuca sativa) , sunflower (Helianthus annuus), safflower

(Carthamus tinctoris), artichoke (Cynara scolymus), burdock

(Arctium lappa), chrysanthemum (Chrysanthemum morifolium) , cosmos (Cosmos bipinnatus), fern-leaf yarrow (Achilla filpendulina) , pot marigold (Calendula arvensis), daisy

(Rudbeckia hirta) , and common zinnia (Zinnia elegans Jasq.).

A transformed composite plant, inter alia, lettuce can be grown by raising outdoors or hydroponics under the known per se conditions. The thus produced transformed composite plant, particularly lettuce can highly express a protein encoded by a gene introduced into a vector. In addition, a transformant of the present invention has an advantage that flying of an introduced gene into environment via pollens can be prevented.

Procedures of the aforementioned genetic engineering or biotechnology can be easily performed according to a method described in commercially available experimental books, for example. Molecular Cloning, Cold Spring Harbor Laboratory published in 1982, and Molecular Cloning, 2nd ed. Cold Spring Harbor Laboratory published in 1989.

The following specific Examples further illustrate the present invention in detail, but the present invention is not particularly limited to them.

Example 1

Sequencing and analysis of lettuce chloroplast genome nucleotide sequence

A lettuce chloroplast is isolated from a lettuce living leaf by percol density gradient centrifugation according to a conventional method. A genome DNA of a lettuce chloroplast is purified from the isolated chloroplast by aDNApurifyingmethod using CTAB (cetyl trimethyl ammonium bromide). A lettuce chloroplast genome library having DNA fragments of about 2kb derived from a chloroplast DNA is prepared from a purified DNA. A nucleotide sequence of 5,428 clones of a genome library was read to sequence a lettuce chloroplast whole genome nucleotide sequence (152,765 bp) . nucleotide sequence information of rbcL (SEQ ID NO: 1 of Sequence Listing) and accD (SEQ ID NO: 2 of Sequence Listing) and nucleotide sequence information of Prrn (SEQ ID NO: 3 of Sequence Listing), and TpsbA (SEQ ID NO: 4 of Sequence Listing) of a lettuce chloroplast genome were obtained from the homology with a tobacco chloroplast genome nucleotide sequence which has been already clarified.

Example 2

Construction of plasmid vector for lettuce chloroplast transformation

(1) Isolation of lettuce chloroplast genome Prrn and TpsbA Prrn of a lettuce chloroplast genome was amplified using a forward primer:

5 ' -CCGCGGCCGCGATATTTTGATTTGCTACCC-S ' (SEQ ID NO: 5 of Sequence Listing) , and a reverse primer:

5'-CCAGCGCTATTCGCCCGGAGTTCGCTCC-3' (SEQ ID NO: 6 of Sequence

Listing) .

The forward primer contains a NotI site at the 5' end, and the reverse primer contains an Eco47III site at a 5 ' end. An amplified fragment was introduced into a Notl and Eco47III site of pLD6 (SEQ ID NO: 17 of Sequence Listing) using restriction enzymes Notl and Eco47III. By the present procedure, a region of nucleotides 2220 to 2345 of a sequence described in SEQ ID NO: 17 of pLD6 was substitutedwithPrrn (SEQ IDNO: 3 of Sequence

Listing: 113 bp) derived from a lettuce chloroplast genome. A terminator TpsbA of psbA of a lettuce chloroplast genome was amplified using a forward primer: 5'-GGCTGCAGGACTTTGGTCTTATTGTAAT-S' (SEQ ID NO: 7 of Sequence Listing) , and a reverse primer:

5'-CCGTCGACGAGCATATTATTTCTTTCTT-S' (SEQ ID NO: 8 of Sequence Listing) .

The forward primer contains a Pstl site at the 5' end, and the reverse primer contains a Sail site at the 5' end. An amplified fragment was introduced into a Pstl and Sail site of pLD6 using restriction enzymes Pstl and Sail. By the present procedure, a region of nucleotides 3174 to 3913 of a sequence described in SEQ ID NO: 17 of pLD6 was substituted with TpsbA (SEQ ID NO: 4 of Sequence Listing; 339 bp) derived from a lettuce chloroplast genome.

Like this, a promoter sequence and a terminator sequence derived from a tobacco of pLD6 were removed to make a plasmid pRL6 in which those sequences were substituted with nucleotide sequences derived from a lettuce chloroplast genome.

As an expression protein, by remaining a spectinomycin resistant gene (hereinafter— abbreviated as aadA: SEQ ID NO: 19 of Sequence Listing) as it is which had been introduced into pLD6 at an initial stage, an expression protein of the present invention was obtained. (2) Isolation of rbcL gene and accD gene

A 1640 bp region containing a rbcL gene of a lettuce chloroplast genome was amplified using a forward primer:

5'-CCGAATTCAATTCATGAGTTGTAGGGAG-S' (SEQ ID NO: 9 of Sequence

Listing) , and a reverse primer: 5'-CCGCGGCCGCGATCCAACCAACACAAAAAT-S' (SEQ ID NO: 10 of Sequence Listing) .

The forward primer contains an EcoRI site at the 5' end, and a reverse primer contains a NotI site at the 5' end. An amplified fragment was introduced into an EcoRI and NotI site of pLD200 (SEQ ID NO: 18 of Sequence Listing) using restriction enzymes EcoRI and Notl. By the present procedure, a region of nucleotides 396 to 2126 of a sequence described in SEQ ID NO: 18 of pLD200 was substituted with rbcL (SEQ ID NO: 1 of Sequence Listing; 1640 bp) derived from a lettuce chloroplast genome. A 1057 bp region containing an accD gene of a lettuce chloroplast genome was amplified using a forward primer: 5'-CCGTCGACGATCCTTAGGATTGGGATAT-S' (SEQ ID NO: 11 of Sequence Listing) , and a reverse primer: 5'-GGAAGCTTCCCATATGAGTAGAACTTTC-S' (SEQ ID NO: 12 of Sequence Listing) .

The forward primer contains a Sail site at the 5' end, and the reverse primer contains a HindIII site at the 5' end. An amplified fragment was introduced into a Sail and HindIII site of pLD200 using restriction enzymes Sail and HindIII. By the present procedure, a region of nucleotides 2141 to 3342 of a sequence described in SEQ ID NO: 18 of pLD200 was substituted with accD (SEQ ID NO: 2 of Sequence Listing; 1057 bp) derived from a lettuce chloroplast genome. Like this, sequences encoding rbcL and accD derived from a tobacco of pLD200 were removed to make a plasmid pRL200 in which those sequences were substituted with nucleotide sequences encoding rbcL and accD derived from a lettuce chloroplast genome.

(3) Preparation of vector for lettuce chloroplast transformation

A DNA fragment (region corresponding to nucleotides 2220 to 3913 of a sequence described in SEQ ID NO: 17) necessary for expressing an aadA gene was excised from the plasmid of the above (1) in which a promoter sequence and a terminator sequence derived from a lettuce chloroplast genome had been introduced, using restriction enzymes Notl and Sail. The excised DNA fragment was introduced into a Notl and Sail site of the plasmid of the above (2) in which rbcL and accD genes derived from a lettuce chloroplast genome had been introduced, using restriction enzymes Notl and Sail. A region into which the DNA fragment is introduced is a region corresponding to nucleotides 2127 to 2141 of a sequence described in SEQ ID NO: 18.

Like this, a vector pRLIOOO for lettuce chloroplast transformation which generates aadA gene expression and homologous recombination with a region of rbcL and accD genes of a lettuce chloroplast genome was prepared (Fig. 1).

Example 3

Transformation and redifferentiation of lettuce chloroplast

After seeding on a MS medium, a living leaf of lettuce which has been cultivated under sterile conditions for about 3 to 4 weeks is used in gene introduction. The MS medium is constructed of 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) , and was adjusted to a pH 5.8 with KOH. Five plates on which 6 to 7 living leaves of lettuce are placed on RMOP medium are prepared, and pre-cultured on the medium for 1 day. A composition of the RMOP medium is such that BA (0.1 mg/L) and NAA (0.1 mg/L) are added to the MS medium. 2.3 mg of a gold particle having a diameter of 0.6 micron, 25 μg of a vector for lettuce chloroplast transformation, 2.5 M calcium chloride and 0.1 M spermidine are mixed, and suspended every minute at 4°C for 10 minutes. After washed with ethanol two times, this is suspended in 60 μL of ethanol. This corresponds to an amount which can be used in an experiment for 10 shooting experiments. Agoldparticle is shot into aleafcellof lettuce using a particle gun (Model PDS-1000/He manufactured by BIO-RAD), for the pre-cultured lettuce leaf. Five shootings are performed per experiment. A shooting pressure is 900 psi. Two days after shooting, a living leaf into which a vector had been shot was cut into a 4 mm square strip with a scalpel, and was placed on RMOP medium containing spectinomyσin (50 mg/L) andpolyvinylpyrrolidone (500 mg/L) . After about 3 to 4 weeks, a green callus was produced from a whitened leaf strip and, thereafter, redifferentiated to reproduce a plant individual.

Example 4

Analysis of lettuce chloroplast transformant by PCR About 50 mg of a leaf tissue of a lettuce plant reproduced in Example 3 was taken, and crushed under liquid nitrogen. To the crushed sample was added with a DNA extraction buffer (0.3 M sodium chloride, 0.05 M Tris-HCl, pH 7.5, 20 mM EDTA, 0.5% SDS, 5 M urea, 5% phenol) , and the materials were mixed. This was extracted with phenol/chloroform (1/1(V/V)), precipitated with ethanol, and air-dried. The extracted DNA was dissolved in 100 μL of a TE solution (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) . In PCR for confirming the presence or the absence of an aadA sequence, a PCR reaction was performedusing a DNA solution extracted from a lettuce plant as a template, andusing a forward primer: 5' -ATGGCTCGTGAAGCGGTTAT-S' (SEQ ID NO: 13 of Sequence Listing) and a reverse primer:

5' -TTATTTGCCAACTACCTTAG-S' (SEQ ID NO: 14 of Sequence Listing) , and the presence or the absence was confirmed by agarose gel electrophoresis. As a control, the pRL 1000 vector prepared in Example 2 anda leaf tissue ofwild strain lettucewere treated similarly. Results are shown in Fig. 2. A 0.8 kb band showing the presence of an aadA genewas recognized in the pRLIOOO vector and the transformed lettuce.

In addition, in order to study the existence ratio of a chloroplast genome with a gene introduced there in a tissue of transformed lettuce and a chloroplast genome with no gene introduced therein which remains a wild type, PCR was performed using an extracted DNA solution of each sample as a template, and using a forward primer: 5'-AGGATTGAGCCGAATCCAAC-S' (SEQ ID NO: 15 of Sequence Listing) and a reverse primer:

5' -AGGATTTGTTCTCTCCTACG-S' (SEQ ID NO: 16 of Sequence listing) .

As a control, a pRL200 vector prepared in Example 2 and a genome DNA extracted from a leaf tissue of wild strain lettuce were treated similarly. Results are shown in Fig.3. In a lane of electrophoresis of a product obtained by PCR using a pRLIOOO vector (lane A) and a genome DNA (lane C) extracted from transformed lettuce as a template, a band showing introduction of a gene into a 1.6kb chloroplast genome could be confirmed. Since a 0.3 kb band showing the presence of a wild type chloroplast genome was recognized in a lane C, although slight, it was recognized that a wild type chloroplast genome DNA was also contained at such an extent of a minor amount that it can be detected by PCR in transformed lettuce (Fig. 3).

Example 5

Assessment of spectinomycin resistance of transformed lettuce In order to investigate whether an aadA gene is expressed, and functions in a lettuce plant for which introduction of an aadA gene was confirmed in Example 4, a 4 mm square leaf strip was excised from transformed lettuce (Fig. 4), and placed on RMOP medium containing 50 mg/L of spectinomycin. Similarly a leaf strip of wild strain lettuce was also placed thereon, and appearance of redifferentiationwas observedat 25°C for 4 weeks under long-day conditions (16 hours light term, 8 hours dark term) (Fig.5) . A, B and C show that a leaf strip of wild strain lettucewas cultured on aRMOPmedium containing spectinomycin. A indicates the appearance of the leaf strip immediately after placing, B indicates the appearance after 2 weeks fromplacing, and C indicates the appearance after 4 weeks, and differentiation in a leaf and a root was not observed in any cases. Similarly, D, E and F show culturing of a leaf strip of transformed lettuce. In E and F, differentiation of a leaf and a root from a leaf strip was confirmed. This indicates that an aadA gene was introduced into a chloroplast genome of lettuce, and an aadA product expressed in a leaf showed resistance to speσtinomycin.

Industrial Applicability

According to the present invention, a medical high value-added protein can be produced safely and at a low cost in a chloroplast of a composite plant in particular, a lettuce.

Claims

1. A vector for transforming a composite plant chloroplast, which comprises aDNA derived from a composite plant chloroplast genome.

2. The vector according to claim 1, wherein the DNA derived from a composite plant chloroplast genome is a DNA derived from a lettuce chloroplast.

3. The vector according to claim 1 or 2, wherein the composite plant is a lettuce.

4. The vector according to any one of claims 1 to 3, which contains a plurality of DNAs derived from a composite plant chloroplast genome.

5. The vector according to claim 4, wherein the DNA derived from a lettuce chloroplast genome is a ribulose-1,5- bisphosphate carboxylase/oxygenase large subunit gene and an acetyl CoA carboxylase subunit gene.

6. The vector according to any one of claims 1 to 5, which comprises at least one kind of a restriction enzyme cleavage site between DNAs derived from the composite plant chloroplast genome.

7. The vector according to any one of claims 1 to 6, which has apromoter and a terminator functioning in a chloroplast between DNA sequences derived from a composite plant chloroplast genome, and has a nucleotide sequence encoding an expression protein between the promoter and the terminator.

8. The vector according to claim 7, wherein the promoter is a promoter of rRNA operon derived from a lettuce chloroplast genome.

9. A vector having a promoter of rRNA operon derived from a lettuce chloroplast genome and a terminator of a psbA gene between a ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit gene and an acetyl CoA carboxylase subunit gene derived from a lettuce chloroplast genome, and having a nucleotide sequence encoding an expression protein between the promoter and the terminator.

10. A composite plant, which is transformed using a vector as defined in any one of claims 1 to 9.

11. A lettuce, which is transformed using a vector as defined in any one of claims 1 to 9.

12. A method of transforming a composite plant chloroplast, which comprises introducing a vector as defined in any one of claims 1 to 9 into a leaf cell of a composite plant.

13. The transformation method according to claim 12, wherein a vector is introduced using a particle gun.

14. A process for producing a transformed plant, which comprises introducing a vector as defined in any one of claims 1 to 9 into a leaf cell of a composite plant, and culturing the leaf cell of a composite plant on a plant culture medium.

15. The process for producing a transformed plant according to claim 14, wherein the plant culture medium contains 0.01 to 1 mg/L of an auxin selected from naphthaleneacetic acid, 2-naphthoxyacetic acid, indoleacetic acid, 4-chloroindoleacetic acid, indolebutyric acid and 2,4-dichlorophenoxyacetic acid and 0.01 to 1 mg/L of a cytokinin selected from kinetin, zeatin, benzyladenine and isopentenyladenine.

16. The process for producing a transformed plant according to claim 15, wherein the weight ratio of cytokinin relative to auxin is 1 : 0.8 to 1.2.

17. The process for producing a transformed plant according to claim 15 or 16, wherein the plant culture medium further contains 100 to 1000 ppm of polyvinylpyrrolidone.

18. A medium for culturing a leaf of a transformed composite plant, which comprises 0.01 to 1 mg/L of an auxin selected from naphthaleneacetic acid, 2-naphthoxyacetic acid, indoleacetic acid, 4-chloroindoleacetic acid, indolebutyric acid and 2,4-diσhlorophenoxyacetic acid, 0.01 to 1 mg/L of a cytokinin selected from kinetin, zeatin, benzyladenine and isopentenyladenine, and 100 to 1000 ppm of polyvinylpyrrolidone.

19. The medium according to claim 18, wherein the weight ratio of cytokinin relative to auxin is 1 : 0.8 to 1.2.

20. The medium according to claim 18 or 19, wherein the composite plant is a lettuce.