JP7094494B2 - Transformed plant of the genus Catharanthus in the family Apocynaceae - Google Patents

Description

The present invention relates to a transformed plant of the genus Catharanthus in the family Apocynaceae. In particular, it relates to a transformed plant of the genus Catharanthus of the Apocynaceae family, which has an increased alkaloid content or production as compared with a wild strain.

Vinca alkaloids such as vinblastine and vincristine are monoterpene indole alkaloids (MIA) extracted from plants of the genus Catharanthus in the family Apocynaceae, and are known to exhibit strong microtube polymerization inhibitory activity. It is also marketed as an anticancer agent for Hodgkin's disease, malignant lymphoma, etc.

Plants of the genus Catharanthus in the family Apocynaceae grow faster than trees and have less environmental impact, so they can be said to be excellent as raw materials for the production of MIA. However, since the amount of alkaloids obtained by extracting plants of the genus Catharanthus of the Apocynaceae family is very small (about 100 μg / gFW in the case of vinca alkaloids), it will meet the expected increase in demand unless the plants are cultivated in large quantities. I can't.

As a method for increasing the production of alkaloids in plants of the genus Catharanthus in the family Apocynaceae, Non-Patent Document 1 describes an anthranilate synthase α subunit having resistance to feedback inhibition by tryptophan derived from arabidopsis as a glucocorticoid-inducible promoter. It is described that the expression in the hair root of Madagascar periwinkle increases the yield of tryptophan and tryptamine, thereby increasing the amount of lochnericine, which is a kind of monoterpene indole alkaloid (MIA). There is.
In addition, Non-Patent Document 2 describes that overexpression of ORCA4 increases the accumulation amount of vinca alkaloid biosynthetic intermediates in madagascar periwinkle roots (page 1108, right column, lines 6 to 7).

Hughes et al., Expression of a Feedback-Resistant Anthranilate Synthase in Catharanthus roseus Hairy Roots Provides Evidence for Tight Regulation of Terpenoid Indole Alkaloid Levels, Biotechnology and Bioengineering, Vol. 86, No. 6, June 20, 2004, pp. 718- 727 Paul et al., A differentially regulated AP2 / ERF transcription factor gene cluster acts downstream of a MAP kinase cascade to modulate terpenoid indole alkanoid biosynthesis in Catharanthus roseus, New Phytologist (2017) 213: pp. 1107-1123

However, in any of the methods of Non-Patent Documents 1 and 2, the expression of the introduced gene is limited to one generation, and there is a big problem that the seeds of the progeny cannot be obtained and propagated in large quantities. In addition, the method of Non-Patent Document 1 has a problem that the expression is limited to hairy roots, it can grow only in a sterile environment, and the amount of indole alkaloids other than locnericin does not increase significantly (non-patent). Patent Document 1 Abstract). Further, the method of Non-Patent Document 2 is also a finding limited to hairy roots as in Non-Patent Document 1, and although the production efficiency of alkaloids is improved to some extent by overexpression of ORCA4, the expected increase in demand in the future is also expected. Considering the above, its production efficiency is not yet sufficient.
The present invention has been made in view of the above problems, and an object of the present invention is to obtain a transformed plant (transgenic plant) in which a foreign gene is incorporated into the genome of a plant belonging to the genus Catharanthus in the family Apocynaceae. It is a thing.
Another object of the present invention is to provide a transformed plant (transgenic plant) capable of producing alkaloids with high efficiency.

As a result of diligent efforts, the present inventors have succeeded in obtaining a transformed plant in which a foreign gene is integrated into the genome of a plant belonging to the genus Catharanthus in the family Apocynaceae.
In addition, as a result of diligent efforts, the present inventors have achieved high efficiency by using a transformed plant in which a gene encoding a protein involved in tryptophan biosynthesis is integrated into the genome of a plant belonging to the genus Catharanthus in the family Apocynaceae. We have found that it is possible to generate alkaloids.
The present invention includes the following configurations.
[1] A transformed plant in which a foreign gene is integrated into the genome of a plant belonging to the genus Catharanthus in the family Apocynaceae.
[2] The transformed plant according to [1], wherein the foreign gene is stably expressed.
[3] The transformation according to [1] or [2], wherein the foreign gene is a foreign gene integrated into the genome of the plant using Agrobacterium rhizogenes and / or melopenem. Plant body.
[4] The transformed plant according to any one of [1] to [3], wherein the Agrobacterium resogenes is Agrobacterium rezogenes A13 strain (MAFF210266).
[5] The transformed plant according to any one of [1] to [4], wherein the exogenous gene is a gene encoding a protein involved in tryptophan biosynthesis.
[6] The transformed plant according to [5], wherein the foreign gene is a gene encoding a protein involved in the biosynthesis of tryptophan derived from Escherichia coli.
[7] The content of at least one alkaloid per unit mass of the transformed plant is 1.1 times higher than the content of at least one alkaloid per unit mass of a wild plant strain of the genus Catharanthus in the family Apocynaceae. The transformed plant according to [5] or [6], which is more than that.
[8] The transformed plant according to [7], wherein the alkaloid is at least one selected from the group consisting of catharanthine, bindrin, and ajmalicine.
[9] The transformed plant according to [7], wherein the alkaloid is catharanthine.
[10] The item according to any one of [5] to [9], wherein the gene encoding the protein involved in the biosynthesis of tryptophan contains at least one selected from the group consisting of aroG, trpD and trpE. Transformed plant body.
[11] At least, the gene encoding the protein involved in the biosynthesis of tryptophan is at least.
The transformed plant according to any one of [5] to [10], which comprises (1) aroG or (2) a combination of trpD and trpE.
[12] The transformed plant according to any one of [5] to [11], wherein the gene encoding the protein involved in the biosynthesis of tryptophan contains aroG, trpD and trpE.
[13] The aroG is a gene modified to suppress the feedback control of 3-deoxy-D-arabino-hepturosonic acid-7-phosphate synthase by phenylalanine, and / or.
The transformed plant according to [10] to [12], wherein trpE is a gene modified so as to suppress feedback control of anthranilic acid synthase by tryptophan.
[14] The aroG is a gene having the DNA sequence of SEQ ID NO: 21, and / or the trpD is a gene having the DNA sequence of SEQ ID NO: 22, and / or the trpE is the DNA of SEQ ID NO: 24. The transformed plant according to any one of [10] to [13], which is a gene having a sequence.
[15] The transformed plant according to any one of [1] to [14], wherein the plant belonging to the genus Catharanthus in the family Apocynaceae is Madagascar periwinkle.
[16] A plant piece obtained from the transformed plant according to any one of [1] to [15].
[17] A step of introducing a foreign gene into a plant belonging to the genus Catharanthus in the family Apocynaceae,
When the plant of the Apocynaceae genus Apocynaceae into which the foreign gene has been introduced is not a callus, a step of forming a callus from the plant into which the foreign gene has been introduced, and a step of differentiating roots and shoots from the callus to obtain a transformed plant. The method for producing a transformed plant according to any one of [1] to [15], which comprises.
[18] The production method according to [17], wherein the foreign gene is a gene encoding a protein involved in tryptophan biosynthesis.
[19] A step of introducing a gene encoding a protein involved in tryptophan biosynthesis into a plant belonging to the genus Catharanthus of the Apocynaceae family.
When the plant of the genus Catharanthus in the family Apocynaceae into which the foreign gene has been introduced is not a callus, a step of forming a callus from the plant into which the foreign gene has been introduced, and a step of differentiating roots and shoots from the callus to obtain a transformed plant. How to make alkaloids, including.
[20] The foreign gene is introduced into a plant belonging to the genus Catharanthus of the Apocynaceae family using Agrobacterium rhizogenes, and roots and shoots are differentiated from callus using a medium containing melopenem, [17] to The production method according to any one of [19].

According to the present invention, it is possible to provide a transformed plant in which a foreign gene is integrated into the genome of a plant belonging to the genus Catharanthus in the family Apocynaceae. Further, according to the present invention, it is possible to provide a transformed plant capable of producing alkaloids with high efficiency.

FIG. 1 is a diagram illustrating an alkaloid synthesis pathway in Madagascar periwinkle. 2 (a) is a design drawing of each vector used in the embodiment of the present invention, and FIG. 2 (b) shows the vector shown in FIG. 2 (a) as a plurality of leaflets of Madagascar periwinkle (TG. 1-33). ), It is a figure which shows the result of having confirmed whether or not the NPTII gene present in the vector was incorporated into the genome of Madagascar periwinkle by genome PCR analysis using the NPTII gene-specific primer. Plants with confirmed bands mean that at least the NPTII gene has been incorporated into the genome of Madagascar periwinkle. In addition, in Examples 1 to 10, TG. 1, TG. 8, TG. 10, TG. 17, TG. 25, TG. 7, TG. 13, TG. 2. TG. 6, TG. 33. FIG. 3 is a diagram showing the free tryptophan content (tryptophan content per unit mass (μmol)) contained in the leaves of Madagascar periwinkle and the leaves of Examples 1 to 5 (n = 3, t-test. * Is P. <0.05, ** means P <0.01). FIG. 4 is a diagram showing the free tryptophan content (tryptophan content per unit mass (μmol)) contained in the leaves of Madagascar periwinkle and the leaves of Examples 6 to 10 (n = 3, t-test. * Is P. <0.05, ** means P <0.01). FIG. 5 is a diagram showing the alkaloid (catharanthine, bindrin, ajmalicine) content (content per unit mass (μg)) contained in the leaves of Madagascar periwinkle and Examples 1 to 5 (n = 3, t-test). * Means P <0.05, ** means P <0.01). FIG. 6 is a diagram showing the alkaloid (catharanthine, bindrin, ajmalicine) content (content per unit mass (μg)) contained in the leaves of Madagascar periwinkle and Examples 6 to 10 (n = 3, t-test). * Means P <0.05, ** means P <0.01). FIG. 7 is a DNA sequence of the aroG gene derived from Escherichia coli. FIG. 8 is a DNA sequence of the aroG4 gene obtained by modifying the aroG gene derived from Escherichia coli. FIG. 9 is a DNA sequence of the trpD gene derived from Escherichia coli. FIG. 10 is a DNA sequence of the trpE gene derived from Escherichia coli. FIG. 11 is a DNA sequence of the trpE8 gene obtained by modifying the trpE gene derived from Escherichia coli. FIG. 12 is a DNA sequence of a chloroplast (chloroplast) transfer signal of aldolase derived from Madagascar periwinkle. FIG. 13 is a DNA sequence of the Nt-JAT2 gene derived from tobacco.

<< I. First aspect >>
The first aspect of the present invention is a transformed plant in which a foreign gene is integrated into the genome of a plant belonging to the genus Catharanthus in the family Apocynaceae. Preferably, it is a transformed plant in which a foreign gene is introduced into a plant belonging to the genus Catharanthus of the Apocynaceae family and the foreign gene is integrated into the genome of the plant.
As used herein and in the claims, "transformed plant" refers to an individual plant whose genetic trait has changed due to the expression of a foreign gene integrated into the plant genome. Therefore, an individual plant in which the introduced foreign gene is not integrated into the plant genome is not included in the transformed plant of the present invention. Further, a plant piece in which a foreign gene is simply introduced into leaves, stems, roots, etc. is not included in the transformed plant of the present invention because it is not an individual plant.
Since the transformed plant of the present invention is an individual plant, it has fertility (sexual fertility) and has an excellent advantage that the foreign gene integrated into the plant genome is passed on to the next generation.
On the other hand, in plant individuals in which the foreign gene is not integrated into the plant genome, the foreign gene is not passed on to the next generation. Further, even a plant piece in which a foreign gene is introduced into a leaf, a stem, a root, or the like does not have fertility, so that the foreign gene cannot be passed on to the next generation.
In the present specification and the scope of the patent claim, the "foreign gene" integrated into the plant genome is a gene different from the gene endogenous to the plant, or the same gene as the gene endogenous to the plant, but outside the plant. A gene introduced from. The foreign gene is preferably a gene different from the gene inherent in the plant, and more preferably a gene derived from an organism (plant, animal, microorganism, etc.) different from the plant. Here, the fact that the foreign gene is the same as the gene endogenous to the plant means that the amino acid sequence of the protein obtained by the expression of the foreign gene is the same as the amino acid sequence of the protein obtained by the expression of the gene endogenous to the integrated plant. Refers to something. The term "foreign gene is different from the gene endogenous to the plant" means that the amino acid sequence of the protein obtained by the expression of the foreign gene is different from the amino acid sequence of the protein obtained by the expression of the gene endogenous to the integrated plant.

The transformed plant of the present invention preferably has a foreign gene integrated into the genome of the plant using Agrobacterium rhizogenes and / or melopenem. Agrobacterium risogenes is a type of bacterium (gram-negative bacterium) belonging to the genus Agrobacterium (genus Rhizobium). The transformed plant of the present invention can be obtained, for example, by infecting a plant of the genus Catharanthus of the Apocynaceae family with a bacterium having a vector containing a foreign gene, and by using Agrobacterium lysogenes as the bacterium. It becomes easy to obtain a transformed plant in which a foreign gene is stably expressed. As the Agrobacterium resogenes bacterium, it is preferable that the Agrobacterium resogenes A13 strain (a strain (wild strain) deposited under the Agricultural Bioresource Gene Bank under the registration number MAFF210266).
Meropenem is a kind of carbapenem antibiotic preparation having antibacterial activity. By using meropenem, it becomes easy to obtain a transformed plant in which a foreign gene is stably expressed. Although the reason is not clear, the use of melopenem can moderately suppress the growth of bacteria after infecting a plant of the genus Catharanthus of the Apocynaceae family with a bacterium containing an exogenous gene, and further, the effect on the growth of the plant. Since there are few plants, it is speculated that the growth of plants is vigorous and the rate of plant mortality due to bacterial growth is reduced.

The foreign gene is not particularly limited, and is a gene encoding a protein involved in tryptophan biosynthesis, a gene encoding a protein involved in the isoprenoid biosynthesis system, a gene encoding a protein involved in binca alkaloid biosynthesis, and further. , Transcription factors that regulate these metabolic systems, and the like. Among these, a gene encoding a protein involved in tryptophan biosynthesis is preferable. That is, the first aspect of the present invention is preferably a transformed plant in which a gene encoding a protein involved in tryptophan biosynthesis is integrated into the genome of a plant belonging to the genus Catharanthus in the family Apocynaceae.

The biosynthetic pathway of tryptophan (L-tryptophan) in the plant body will be described. L-Tryptophan is biosynthesized from chorismic acid, a common intermediate in aromatic amino acids. L-tryptophan is biosynthesized by the action of five enzymes synthesized by the tryptophan operon trpEDCBA on chorismic acid produced via a joint shikimate pathway starting with phosphoenolpyruvate and D-erythrose-4-phosphate. Will be done. First, anthranilic acid synthase (TrpE-TrpD polymer) acts on chorismic acid to synthesize anthranilic acid, and then anthranilic acid phosphoribosyltransferase (TrpD) acts to synthesize anthranilic acid. Then, phosphorribosyl anthranilic acid isomerization enzyme (TrpC) and indole-3-glycerol phosphate synthase (TrpC) act to synthesize indole-3-glycerol phosphate, which is here with tryptophan synthase (TrpB-TrpA weight). L-tryptophan synthesis is completed (Bonggaerts et al., MetabEng, 3,289-300, 2001).
As described above, various proteins (enzymes) are involved in the biosynthesis of tryptophan, and the amount of tryptophan synthesized can be increased by increasing the amount of these enzymes in the living body.
Further, it is known that when the amount of tryptophan synthesized increases, tryptophan binds to the α subunit (TrpE) of anthranilic acid synthase to perform negative feedback control. Therefore, the amount of tryptophan synthesized can be further increased by modifying the α subunit (trpE) of the anthranilic acid synthase so that it cannot bind to tryptophan.

<1. Genes encoding proteins involved in tryptophan biosynthesis>
Genes encoding proteins involved in tryptophan biosynthesis include anthranilic acid synthase (trpE), phosphoglycerate dehydrogenase (serA), and 3-deoxy-D-arabinohepturonic acid-7-phosphate synthase ( aroG), 3-dehydrokinate synthase (aroB), shikimic acid dehydrogenase (aroE), shikimic acid kinase (aroL), 5-enoylpyrrovinyl shikimic acid-3-phosphate synthase (aroA), chorismate synthesis Enzyme (aroC), prefenate dehydratase, chorismate mutase (pheA) and tryptophan synthase (trpA, trpB), phosphoribosyl anthranilic acid isomerase (TrpC), indol-3-glycerol phosphate synthase (TrpC) anthranyl acid Examples include the gene encoding the phosphoribosyl transferase (TrpD). In addition, these genes may be modified so that the enzyme activity of the protein (enzyme) encoded by the gene becomes large. In addition, it is known that it receives negative feedback control by binding to tryptophan and α-subunit (trpE) of anthranyl acid synthase, which is known to receive negative feedback control, and it is known to receive negative feedback control by binding to phenylalanine. The gene encoding deoxy-D-arabinohepturonic acid-7-phosphate synthase (aroG) has been modified to reduce or release the negative feedback control. It is also good. The gene encoding the protein involved in tryptophan biosynthesis may be one type of gene or two or more types of genes.

Among these, the gene encoding the protein involved in tryptophan biosynthesis preferably contains at least one selected from the aroG, trpD and trpE genes, and is preferably a combination of the trpD gene and the trpE gene and / or the aroG gene. Is more preferably included, and even more preferably to include the aroG, trpD and trpE genes. Further, the trpE gene may be a trpE gene modified so as to suppress the feedback control of anthranilic acid synthase by tryptophan. The aroG gene may be an aroG gene modified to suppress the feedback control of 3-deoxy-D-arabino-hepturosonic acid-7-phosphate synthase by phenylalanine.
The gene encoding the protein involved in tryptophan biosynthesis is preferably the gene derived from Escherichia coli.
Hereinafter, the aroG, trpD and trpE genes will be described.

(AroG gene)
aroG is an enzyme involved in the synthesis of 3-deoxy-D-arabinohepturonic acid 7 phosphate (NAHP), the first precursor of aromatic amino acids during tryptophan biosynthesis, that is, 3-deoxy-7-phosphohepturonic acid. It is a gene encoding a synthase (AroG). By the action of the enzyme, NAHP is synthesized from phosphoenolpyruvate and D-erythrose 4-phosphate, and NAHP is metabolized to chorismic acid through several metabolic processes, and finally tryptophan biosynthesis. The amount increases. Examples of organisms having a gene (aroG) encoding the enzyme correspond to the majority of plants and microorganisms, and examples thereof include E. coli, Corynebacterium glutamicum, Bacilus subtils, Arabidopsis thaliana, Oryza sativa and the like. In the present invention, the aroG gene may be a plant-derived and / or a microorganism-derived aroG gene. Preferably, the aroG gene is a gene derived from E. coli.
In the present specification and claims, the aroG gene may have a mutation as long as the protein obtained by expressing the gene functions as a 3-deoxy-7-phosphohepturonic acid synthase. The aroG gene preferably has 90% or more homology with the aroG gene possessed by any of the above plants or microorganisms (preferably E. coli), and more preferably 95% or more homology with the sequence. , 98% or more homology with the sequence is preferable, 99% or more homology with the sequence is even more preferable, and 100% homology with the sequence may be obtained.
The mutant gene of aroG may or may not be modified so as to release the feedback control by phenylalanine, but a modified gene is preferable. By using the mutant gene, even if the amount of tryptophan biosynthesis increases, negative feedback control does not occur, so that the amount of tryptophan biosynthesis can be further increased. As such aroG gene, aroG4 gene, aroG15 gene and the like are known. For the aroG4 gene, see APPLIED AND ENVIRONMENTAL MICROBIOROGYFeb. 1997, p. You can refer to 761-762. The aroG15 gene can be referred to in the same manner.
The aroG gene is preferably a gene having a DNA sequence having 90% or more homology with the DNA sequence shown in SEQ ID NO: 20 or 21 derived from Escherichia coli, and a DNA sequence having 95% or more homology with the sequence. It is more preferable that the gene has a DNA sequence having 98% or more homology with the sequence, and it is even more preferable that the gene has a DNA sequence having 99% or more homology with the sequence. It is even more preferable that the gene has a DNA sequence having 99.5% or more homology with the sequence, and the DNA sequence having 99.9% or more homology with the sequence is even more preferable. It is even more preferably a gene having the DNA sequence, further preferably a gene having the DNA sequence shown in SEQ ID NO: 20 or 21, and most preferably a gene having the DNA sequence shown in SEQ ID NO: 21 (aroG4).

(TrpD gene)
trpD is a gene encoding anthranilic acid phosphoribosyltransferase (TrpD). By the action of the enzyme, anthranilic acid and 5-phosphoribosyl diphosphate are synthesized from anthranilic acid and anthranilic acid, and the synthesized anthranilic acid is metabolized through several metabolic processes, and finally the amount of tryptophan biosynthesis is increased. Increase. Examples of organisms having the gene correspond to the majority of plants and microorganisms, and examples thereof include E. coli, Corynebacterium glutamicum, Bacilus subtils, Arabidopsis thaliana, and Oryza sativa. In the present invention, the trpD gene may be a trpD gene derived from a plant and / or a microorganism. Preferably, the trpD gene is a gene derived from E. coli.
As far as the present specification and claims are concerned, the trpD gene may have a mutation as long as the protein obtained by expressing the gene functions as an anthranilic acid phosphoribosyl transferase. The trpD gene preferably has 90% or more homology with the trpD gene possessed by either of the above plants or microorganisms (preferably E. coli), and more preferably 95% or more homology with the sequence. , 98% or more homology with the sequence is preferable, 99% or more homology with the sequence is even more preferable, and 100% homology with the sequence may be obtained.
The trpD gene is preferably a gene having a DNA sequence having 90% or more homology with the DNA sequence shown in SEQ ID NO: 22 derived from Escherichia coli, and has a DNA sequence having 95% or more homology with the sequence. It is more preferably a gene, and even more preferably a gene having a DNA sequence having 98% or more homology with the sequence, and a gene having a DNA sequence having 99% or more homology with the sequence. It is even more preferable that the gene has a DNA sequence having 99.5% or more homology with the sequence, and the gene has a DNA sequence having 99.9% or more homology with the sequence. Is even more preferable, and the gene having the DNA sequence shown in SEQ ID NO: 22 is most preferable.

(TrpE gene)
The trpE gene is a gene encoding the alpha subunit (TrpE) of anthranilate synthase. By the action of the enzyme, anthranilic acid, glutamic acid, and pyruvic acid are synthesized from corismic acid and glutamine, and the synthesized anthranilic acid is metabolized through several metabolic processes, so that the amount of tryptophan biosynthesis is finally increased. Increase. Examples of organisms having the gene correspond to the majority of plants and microorganisms, and examples thereof include E. coli, Corynebacterium glutamicum, Bacilus subtils, Arabidopsis thaliana, and Oryza sativa. In the present invention, the trpE gene may be a trpE gene derived from a plant and / or a microorganism. Preferably, the trpE gene is a gene derived from E. coli.
In the present specification and claims, the trpE gene is used as long as the protein obtained by expressing the gene functions as an α subunit of anthranilate synthase and acts as an anthranilate synthase together with the β subunit. It may have a mutation. The trpE gene preferably has 90% or more homology with the trpE gene possessed by either of the above plants or microorganisms (preferably E. coli), and more preferably 95% or more homology with the sequence. , 98% or more homology with the sequence is preferable, 99% or more homology with the sequence is even more preferable, and 100% homology with the sequence may be obtained.
The above-mentioned mutant gene of trpE may or may not be modified so as to suppress the feedback control of anthranilic acid synthase by tryptophan, but the modified gene is preferable. As such trpE gene, trpE8 gene, TRP4 and the like are known. For the trpE8 gene, WO94 / 08031, No. 7-507693 can be referred to. For the TRP4 gene, THE PLANT CELL 1993 VOL5, p1011-1827 can be referred to.
The trpE gene is preferably a gene having a DNA sequence having 90% or more homology with the DNA sequence shown in SEQ ID NO: 23 or 24 derived from Escherichia coli, and a DNA sequence having 95% or more homology with the sequence. It is more preferable that the gene has a DNA sequence having 98% or more homology with the sequence, and it is even more preferable that the gene has a DNA sequence having 99% or more homology with the sequence. It is even more preferable that the gene has a DNA sequence having 99.5% or more homology with the sequence, and the DNA sequence having 99.9% or more homology with the sequence is even more preferable. It is even more preferably a gene having the DNA sequence, further preferably a gene having the DNA sequence shown in SEQ ID NO: 23 or 24, and most preferably a gene having the DNA sequence shown in SEQ ID NO: 24 (trpE8).

<2. DNA sequence encoding the signal sequence for the transfer of aldolase to chloroplast>
In the transformed plant of the present invention, the gene encoding the protein involved in the biosynthesis of tryptophan integrated into the genome is the DNA encoding the transfer signal sequence of aldolase derived from Nichinichisou to chloroplast (chloroplast). It may be concatenated with an array. The DNA sequence encoding the signal sequence is preferably linked to the 5'end side of each gene encoding a protein involved in tryptophan biosynthesis. Having the signal sequence facilitates the transport of the expressed protein into the chloroplast. Since tryptophan is synthesized in chloroplast, the amount of tryptophan synthesized can be increased by having the signal sequence. The DNA sequence encoding the transfer signal sequence of aldolase to chloroplast preferably contains a DNA sequence having 90% or more homology with the DNA sequence shown in SEQ ID NO: 25, and has 95% or more homology with the sequence. It is more preferable to include a DNA sequence having, further preferably to contain a DNA sequence having 98% or more homology with the sequence, and even more preferably to include a DNA sequence having 99% or more homology with the sequence. It is even more preferable to include a DNA sequence having 99.5% or more homology with the sequence, and even more preferably to contain a DNA sequence having 99.9% or more homology with the sequence. It is even more preferable to include the DNA sequence shown in SEQ ID NO: 25, and most preferably the DNA sequence shown in SEQ ID NO: 25.
Further, it is preferable that the DNA sequence encoding the signal sequence is linked to the 5'end side of each gene encoding a protein involved in tryptophan biosynthesis and has no stop codon. By not having a termination codon, the signal sequence and the protein involved in tryptophan biosynthesis are expressed as one protein, so that the protein involved in tryptophan biosynthesis is transferred to chloroplast through the N-terminal signal sequence. Will be.

<3. Nt-JAT2 gene>
The Nt-JAT2 (Nicotiana tabacum jasmonte-inducible alkaloid transporter 2) gene is a vacuolar-localized alkaloid transporter isolated from cultured tobacco cells and having a function of transporting nicotine alkaloids to vacuoles.
In the transformed plant of the present invention, the Nt-JAT2 gene may be integrated into the plant genome. Further, in the transformed plant of the present invention, the Nt-JAT2 gene may be stably expressed. The Nt-JAT2 gene is preferably a gene derived from tobacco (Nicotiana tabacum). The tobacco-derived Nt-JAT2 gene is preferably a gene having a DNA sequence having 90% or more homology with the DNA sequence shown in SEQ ID NO: 26, and a DNA sequence having 95% or more homology with the sequence. It is more preferable that the gene has a DNA sequence having 98% or more homology with the sequence, and it is even more preferable that the gene has a DNA sequence having 99% or more homology with the sequence. It is even more preferable that the gene has a DNA sequence having 99.5% or more homology with the sequence, and the DNA sequence having 99.9% or more homology with the sequence is even more preferable. It is even more preferable that the gene has the gene, and most preferably the gene has the DNA sequence shown in SEQ ID NO: 26.
Since the stable expression of the Nt-JAT2 gene causes the alkaloids produced in the transformed plant of the present invention to be accumulated and sequestered in the vacuole, the degradation of the alkaloids in the cells can be prevented. ..

<4. Stable expression>
In the present specification and claims, "stable expression" or "stable expression" means that the introduced foreign gene is constitutively expressed. This expression is used to distinguish it from the case of using a transient expression or inducible expression vector system. In the transient expression system or the inducible expression vector system, the temporarily introduced gene is expressed in the plant, but since the expression is short-term, the effect of the foreign gene should be confirmed only in a limited manner. I can't. On the other hand, in the transformed plant of the present invention, since the foreign gene is integrated into the plant genome, the foreign gene can be stably expressed throughout the entire growing period.
In the transformed plant of the present invention, it is preferable that the foreign gene is stably expressed, and it is more preferable that the gene encoding the protein involved in tryptophan biosynthesis is stably expressed. The stable expression of the gene encoding the protein involved in tryptophan biosynthesis makes it possible to stably increase the amount of alkaloids produced from plants over a long period of time.
<5. Tryptophan content>
It is preferable that the transformed plant of the present invention has an increased tryptophan content (or expression level or synthetic amount).
In addition, the transformed plant of the present invention has a tryptophan content per unit mass (or, as compared with a wild strain of a plant belonging to the genus Catharanthus of the Apocynaceae family, in which a gene encoding a protein involved in tryptophan biosynthesis has not been introduced. The expression level or synthesis amount) is preferably 1.5 times or more, more preferably 2 times or more, further preferably 2.5 times or more, and even more preferably 3 times or more. It is preferable, and even more preferably, it is 4 times or more. When the tryptophan content is within the above range, the amount of alkaloids obtained from plants tends to increase.
The degree of tryptophan content can be directly evaluated by confirming the amount of tryptophan contained in the plant or plant site.
In addition, "a plant wild strain of the genus Catharanthus of the Apocynaceae family in which a gene encoding a protein involved in tryptophan biosynthesis has not been introduced" means that a gene encoding a protein involved in tryptophan biosynthesis is introduced from outside the plant. A plant strain that has not been used.

<6. Plants>
The transformed plant of the present invention is a plant belonging to the genus Catharanthus of the family Apocynaceae. Among these, it is more preferable to use Catharanthus roseus.

<7. Alkaloid>
The transformed plant of the present invention synthesizes at least one alkaloid per unit mass as compared with a wild strain of a plant belonging to the genus Catharanthus of the family Apocynaceae, in which a gene encoding a protein involved in tryptophan biosynthesis has not been introduced. The amount is preferably increased, the synthesis amount of at least one alkaloid is more preferably 1.1 times or more, and more preferably 1.2 times or more than that of the wild strain. , 1.3 times or more is further preferable, 1.4 times or more is further preferable, and 1.5 times or more is particularly preferable.
The alkaloids are preferably binca alkaloids (particularly Vinblastine and Vincristine) and at least one alkaloid selected from their biosynthetic intermediates, preferably catharanthine, Vindoline, etc. It is more likely that it is at least one selected from the group consisting of Ajmalicine, Vinblastine, Vincristine, Tabersonine, Serpentine, Stemmadenine and Strictosidine. It is even more preferably at least one selected from the group consisting of catharanthine, Vindoline, Vinblastine, Vincristine, Ajmalicine, and even more preferably catharanthine, bindrin. It is even more preferably at least one selected from the group consisting of (Vindoline) and Ajmalicine, and further preferably at least one selected from the group consisting of Catharanthine and Ajmalicine. More preferably, it is particularly preferably catharanthine.
Tabersonine, Vindoline, Catharanthine, Vinblastine, Vincristine, Ajmalicine, Serpentine, Stemmadenine, Stemmadenine, Stricto That's right.

Vinca alkaloids (vinblastine, vincristine, vincristine, vinorelbine) are known to exhibit strong microtubule polymerization inhibitory activity. It is also known to exert an anticancer drug action against Hodgkin's disease, malignant lymphoma and the like.
Therefore, the transformed plant of the present invention is also useful for producing alkaloids or alkaloid-containing compositions for use in these applications.

The transformed plant of the present invention can stably increase the amount of alkaloids such as catharanthine and bindrin produced over a long period of time. In Non-Patent Document 1, even if the mutant trpE gene encoding anthranilic acid synthase in which the degree of feedback inhibition by tryptophan is weakened is temporarily expressed using a dexamethasone-inducing vector, alkaloids other than the amount of locnericin are used. The effect of the present invention is unexpected and surprising given that the amount did not increase significantly. The reason why the transformed plant of the present invention can increase the production of alkaloids such as catharanthine is not clear, but it is presumed as follows. Catharanthine is synthesized in the epidermis of leaves, and it is thought that part of it binds to bindrin and is used for vinblastine biosynthesis. It is also believed that unused excess catharanthine is released and accumulated in the outermost cuticle layer of the leaf. Unlike the hairy roots of Non-Patent Document 1, the transformed plant of the present invention has leaves and stems and is capable of growth, so that excess catharanthine not used in the process of growth is always gradually lengthened. It is speculated that the catalanthin content may have increased as a result of the accumulation over a period of time.
In addition, since the transformed plant of the present invention incorporates a gene encoding a protein involved in tryptophan biosynthesis, alkaloids can be stably increased over a long period of time, and progeny seeds are used. As a result, it can be easily propagated in large quantities, which is very advantageous in the industry.
<< II. Second aspect >>
The second aspect of the present invention is a plant piece obtained from the transformed plant body of the first aspect. The plant piece is part of the transformed plant of the first aspect. The plant piece may be any of leaves, stems and roots, but leaves and / or stems are preferable, and leaves are more preferable.

<< III. Third aspect and fourth aspect >>
A third aspect of the present invention is a step of introducing a foreign gene into a plant belonging to the genus Catharanthus of the family Apocynaceae. A method for producing a transformed plant, which comprises a step of forming a catharanthus and a step of differentiating roots and shoots (that is, leaves and stems) from callus to obtain a transformed plant.
Preferably, a third aspect of the present invention is a step of introducing a gene encoding a protein involved in tryptophan biosynthesis into a plant of the genus Catharanthus of the family Apocynaceae, and a plant of the genus Catharanthus of the family Apocynaceae into which the gene has been introduced is not a callus. In the case, it is a method for producing a transformed plant, which comprises a step of forming callus from a plant into which the gene has been introduced and a step of differentiating roots and shoots from the callus to obtain a transformed plant.
A fourth aspect of the present invention is a step of introducing a foreign gene into a plant belonging to the genus Catharanthus of the family Apocynaceae. A method for producing an alkaloid, which comprises a step of forming a catharanthus and a step of differentiating roots and shoots from a crow to obtain a transformed plant.
Preferably, a fourth aspect of the present invention is a step of introducing a gene encoding a protein involved in the biosynthesis of tryptophan into a plant of the genus Catharanthus of the family Apocynaceae, in which the plant of the genus Catharanthus of the family Apocynaceae into which the gene is introduced is not a callus. Is a method for producing an alkaloid, which comprises a step of forming a dogbanes from a plant into which the gene has been introduced and a step of differentiating roots and shoots from the catharanthus to obtain a transformed plant.
Examples of foreign genes, genes encoding proteins involved in tryptophan biosynthesis, plants, and the like are as described above.
The plant into which the foreign gene is introduced may be a plant itself, a part of the plant, or a callus, but is preferably a part of the plant or a callus, and is a plant. The part of the body is preferably leaves and / or stems, more preferably leaves. The plant into which the foreign gene is introduced is particularly preferably a leaf or a callus, and most preferably a leaf.

<1. Gene introduction>
The method of the present invention comprises the step of introducing a foreign gene (preferably a gene encoding a protein involved in tryptophan biosynthesis) into a plant belonging to the genus Catharanthus in the family Apocynaceae.
The introduction of the foreign gene is not particularly limited as long as the gene is introduced into the plant, but it is preferable to introduce the foreign gene into a plant belonging to the genus Catharanthus in the family Apocynaceae using a vector containing the foreign gene.

Examples of the method for introducing a foreign gene include an Agrobacterium method and a particle gun method.
The Agrobacterium method is the soil bacterium Rhizobium genus Agrobacterium tumefaciens (current official scientific name: Rhizobium rhadiobacter) and Agrobacterium lizogenes (Agrobacterium). Official scientific name: A method using Rhizobium rhizogenes, which utilizes the fact that the T-DNA region of the Ti or Ri plasmid possessed by this bacterium is inserted into the plant genome by homologous recombination. .. That is, if the target gene is inserted into the T-DNA region, the target gene can be introduced into the plant genome when Agrobacterium infects the plant.
The particle gun method is a method in which fine particles of metal such as gold or tungsten coated with DNA are ejected as bullets at high speed to introduce DNA into plant cells. High-pressure gas such as helium is mainly used for the injection of metal particles.
Among these, it is preferable that the method of the present invention introduces a foreign gene (particularly a gene encoding a protein involved in tryptophan biosynthesis) into a plant belonging to the genus Catharanthus in the family Apocynaceae by the agrobacterium method.

In the agrobacterium method, for example, an agrobacterium containing a vector containing a foreign gene (particularly a gene encoding a protein involved in the biosynthesis of tryptophan) is infected with a leaflet or callus of Nichinichisou to eradicate the agrobacterium. By adding an antibiotic for selecting a gene-introduced cell and an antibiotic for selecting a gene-introduced cell to a medium and repeating eradication and selection, the gene can be introduced into the plant.
Among these, in the method of the present invention, it is preferable to introduce the foreign gene into a plant belonging to the genus Catharanthus of the Apocynaceae family using Agrobacterium rhizogenes. It is more preferable to use the Um-Rhizobium rhizogenes A13 strain (a strain (wild strain) deposited under the registration number MAFF210266 in the Agricultural Bioresource Genebank). By using Agrobacterium retogenes, the probability that callus will be formed from plants after introduction of foreign genes and the probability that roots and shoots will be formed from callus will increase, making it easier to obtain transformed plants. Become.

The vector is not particularly limited as long as the above gene can be expressed in the plant, and various plasmid vectors can be used. For example, pRI201-AN vector (Takara Bio Inc.), pBI vector (Takara Bio Inc.) , PRI vector (Takara Bio Inc.), pFAST vector (Thermo Fisher Scientific), pSuperAgro vector (Implanta Innovations) can be used. Among these, the pRI201-AN vector is preferable.
As a method of incorporating a gene encoding a protein involved in tryptophan biosynthesis into a vector, for example, a method of incorporating by cleaving with a restriction enzyme and connecting by a ligation reaction, a homologous group such as an In-fusion method or a Gibson assembly method, etc. There is a method of incorporating by replacement.

<2. Generation of transformed plants>
In the method of the present invention, when the plant of the genus Catharanthus in the family Apocynaceae into which the foreign gene has been introduced is not a callus, a step of forming a callus from the plant into which the gene has been introduced, and a step of transforming the callus into roots and shoots. It has a step of obtaining a plant. Transformed plants can be obtained by differentiating roots and shoots from callus.
The method of the present invention does not require a step of forming callus when the plant of the genus Catharanthus in the family Apocynaceae into which the foreign gene has been introduced is in the form of callus. On the other hand, when the plant of the genus Catharanthus in the family Apocynaceae into which the foreign gene has been introduced is not callus, for example, when it is a leaf, stem and / or root or plant itself, a step of forming callus from the plant into which the gene has been introduced. Have.

As a method of forming callus from a plant into which a gene has been introduced and / or a method of differentiating root and shoot from callus, a step of differentiating root and shoot from callus, or a step of forming callus from a plant and In both the steps of differentiating roots and shoots from callus, callus-introduced plants or callus (particularly plants or callus infected with agrobacterium having a vector containing the foreign gene) is preferably a medium containing melopenem. Is preferably cultured in a solid medium. The concentration of meropenem in the medium is preferably about 10-50 mg / l, more preferably about 10-30 mg / l, and even more preferably about 20 mg / l. By culturing a plant or callus infected with Agrobacterium in a medium containing melopenem, roots and shoots can be differentiated from the callus, and transformed plants can be easily obtained.
As the medium, for example, 1 / 2B5 medium obtained by diluting Ganborg B5 medium (Wako Pure Chemical Industries, Ltd.) in half is used as the basic medium, and melopenem, 30% sucrose, and MS vitamin are added to adjust the pH to 5.8. After the adjustment, a solid medium solidified by adding 5% gellite (Wako Pure Chemical Industries, Ltd.) can be mentioned. When a plant piece infected with Agrobacterium is placed on this solid medium and cultured at 24 ° C. for 16 hours for 10 days to 2 weeks, callus is formed at the cut end of the plant piece. The leaf pieces in which callus can be confirmed are transplanted to a new medium having the same composition, and when the culture is continued, root regeneration can be confirmed from the callus 2 weeks to 1 month after transplantation. Furthermore, if the leaf pieces in which the root regeneration was observed are continuously transplanted to a new medium under the same conditions every two weeks and the culture is continued, shoots are regenerated from the vicinity of the callus in about 1 to 2 months. To. The obtained shoots are cut out from stems and transplanted to a new medium having the same composition, and the roots are regenerated in about 2 weeks to 1 month to obtain a complete plant.
The transformed plant thus obtained can stably express the gene introduced into later generations.

The method of the present invention is Agrobacterium rhizogenes (preferably, the Agrobacterium rhizogenes bacterium is Agrobacterium rhizogenes A13 strain (a strain deposited as registration number MAFF210266 in the Agricultural Biological Resources Genebank). (Wild strain))) is used to introduce the foreign gene into a plant belonging to the genus Agrobacterium of the family Rhizobium rhizome, and a medium containing melopenem, preferably a solid medium, is used to differentiate roots and shoots from callus (preferably containing melopenem). It is preferable to use a medium to form callus from the plant and to differentiate roots and shoots from the callus). This makes it easier to obtain a transformed plant.

<3. Selection of plants into which genes have been introduced>
The method of the present invention may include a step of introducing a foreign gene (particularly a gene encoding a protein involved in tryptophan biosynthesis) into a plant and then selecting a plant into which the foreign gene has been introduced. This step may be before the production step of the transformed plant, may be simultaneous with the production step of the transformed plant, or may be after the production step of the transformed plant, but may be transformed. It is preferable that the process is before and / or at the same time as the step of forming the transformed plant.
Examples of the method for selecting the plant include a method of introducing a vector containing an arbitrary drug resistance gene together with a foreign gene into the plant and then culturing the plant in a medium containing a drug corresponding to the arbitrary drug resistance gene. Examples of the drug resistance gene include a kanamycin resistance gene, a hygromycin resistance gene, and a bialaphos resistance gene. In addition, examples of the drug corresponding to these resistance genes include kanamycin, hygromycin, and bialaphos. For example, when a neomycine phosphotransphase (NPT) gene is inserted into the T-DNA region of the vector, selection is performed with a medium containing about 50-100 mg / l of the antibiotic canamycin, and hygromycin (hygromycin) ( When the hygromycin resistance gene (HPT) and the bialaphos resistance gene (bar) gene are inserted, 25-50 mg / l hygromycin (Hygromycin) B and 10-25 mg / l Vialaphos, respectively, are added to the medium. By culturing a plant infected with hygromycin in a medium supplemented with a drug corresponding to these drug resistance genes, the plant into which the gene has been introduced can be selected.
Among these, in the method of the present invention, it is preferable to select a medium containing 50-100 mg / l kanamycin and select a plant into which the gene has been introduced.
<4. Selection of transformed plants expressing foreign genes>
The method of the present invention may include a step of selecting a transformed plant in which a foreign gene is expressed after a step of producing a transformed plant (after differentiating shoots and roots). Selection of transformed plants expressing a foreign gene can be confirmed, for example, by measuring the amount of mRNA derived from the foreign gene. It can also be confirmed by measuring the amount of protein derived from a foreign gene. Among these, it is preferable to confirm by measuring the amount of protein derived from a foreign gene.

<5. Extraction>
The method of the present invention may include a step of extracting an alkaloid from the transformed plant after obtaining a transformed plant or selecting a transformed plant expressing a foreign gene. .. As a method for extracting alkaloid from the transformed plant, a method of crushing the leaves of the raw transformed plant and extracting with a solvent such as methanol, or a method of crushing the leaves of the dried transformed plant and using a solvent such as methanol is used. The method of extracting with is mentioned.
In the extraction method from fresh leaves, for example, alkaloids are extracted from transformed plants by freezing them in liquid nitrogen, crushing them, adding 99% methanol, and shaking them at 30 ° C. for about 2 hours. Can be done.
In the extraction method from dried leaves, for example, freeze-dried leaves are crushed with a mixer or the like, 99% methanol is added, and the leaves are shaken at 30 ° C. for about 2 hours to extract alkaloids from the transformed plant. Can be done.

Hereinafter, the usefulness of the present invention will be specifically described with reference to examples. However, the present invention is not limited thereto.

<< 1. Materials and methods >>
(1) Aseptic sowing of Madagascar periwinkle seeds and pre-culture of cotyledons Using seeds of Madagascar periwinkle (variety: Pacifica Takii Seed Co., Ltd.), soak in 70% ethanol for 1 minute, and then 2% sodium hypochlorite (5-fold). Diluted) for 15 minutes. After washing 3 times with sterile water in a clean bench, the seeds were sown on 1/2 B5 agar medium (Ganborg B5 medium). Culturing was carried out at 25 ° C. for 16 hours. The cotyledons of the seedlings 20 days after sowing were cut out so as not to have growth points, placed on the same 1/2 B5 agar medium, and cultured 24 hours before.

(2) Acquisition of a gene encoding a target protein and addition of a signal sequence For each of the aroG gene, trpE gene, and trpD gene, E.I. Obtained from the DNA of colli by the PCR method (aroG: SEQ ID NO: 20, trpE: SEQ ID NO: 23, trpD: SEQ ID NO: 22). All the genes obtained by cloning using the TA cloning kit were subjected to sequence analysis, and it was confirmed that there was no difference from the base sequence information. Using the PrimeSTARMutagenesis Basal Kit (Takara Bio), the 150th amino acid of the amino acid sequence of the aroG gene is mutated from proline (codon sequence: cca) to lysine (codon sequence: cta) to obtain the aroG4 gene. (SEQ ID NO: 21). Similarly, using the PrimeSTARMutagenesis Basic Kit (Takara Bio), the 21st amino acid of the amino acid sequence of the trpE gene is mutated from proline (codon sequence: ccc) to serine (codon sequence: tcc) by the PCR method and 50. The second amino acid was mutated from lysine (codon sequence: aaa) to glutamate (codon sequence: gaa) to give the trpE8 gene (SEQ ID NO: 24). Sequence analysis was also performed on the mutated gene, and it was confirmed that base substitution had occurred.
In plants, tryptophan is synthesized in chloroplasts, so that the protein synthesized in the plant can be transported to chloroplasts, so that the signal sequence for the transfer of aldolase derived from nichinichisou to chloroplasts (GenBank: GU723954). , N-terminal 174bp, SEQ ID NO: 25) was obtained by synthesis and ligated to the N-terminal side of each gene of aroG4, trpE8 and trpD (referred to as Ald-aroG4, Ald-trpE8 and Ald-trpD, respectively). ). For ligation between the signal sequence and aroG4, a primer (SEQ ID NO: 7) prepared on the 5'side of Aldrace, a primer (SEQ ID NO: 9) of the reverse sequence prepared in the ligation region of Aldrace and aroG4, and Aldrase and aroG4. A pair of a primer of the forward sequence (SEQ ID NO: 8) prepared in the linking region of AroG4 and a primer of the reverse sequence (SEQ ID NO: 2) prepared on the 3'side of aroG4, and a signal for transfer of Aldrose to chloroplast. PCR was performed using the gene (SEQ ID NO: 25) and the aroG4 gene (SEQ ID NO: 21) as templates, and then the amplified gene fragments were confirmed to be of the desired size by electrophoresis, and then DNA was removed from the gel. Cut out, purified using Wizard SV Gel and PCR Clean-Up System (Promega), and using a mixture of each gene as a template, the primer (SEQ ID NO: 7) prepared on the 5'side of Aldrose and 3 of aroG4. Each gene was ligated by performing PCR using the reverse sequence primer (SEQ ID NO: 2) prepared on the'side. By the same method, the above signal sequence and trpE8 were ligated using the primers of SEQ ID NOs: 4, 7, 10 and 11. The above signal sequence and the trpD gene were ligated using the primers of SEQ ID NOs: 6, 7, 12, and 13.
For the Nt-JAT2 gene, primers (SEQ ID NOs: 14 and 15) were prepared based on genomic information (GenBank: AB922128), and cDNA prepared from tobacco leaves was used as a template and amplified by PCR. The obtained genes were subjected to sequence analysis and confirmed to have the same base sequence (SEQ ID NO: 26).

(3) Preparation of binary vector for expression The obtained four genes (Ald-aroG4, Ald-trpE8, Ald-trpD, Nt-JAT2) were used as a binary vector and a promoter of pRI201-AN (TAKARA BIO.), Respectively. Inserted between the terminators, these four gene sets are amplified by the PCR method from the upstream of the promoter to the downstream of the terminator using the primers of SEQ ID NOs: 16 and 17, and the Gibson assembly system (NEW ENGLAND BioLabs) is amplified. Using this, each gene set was ligated to complete an expression vector (JAED gene group expression vector). The ligated binary vector was transformed into two types of Agrobacterium (Agrobacterium tumefaciens, LBA4404 and Agrobacterium rhizogenes, A13) and used in the transformation experiment to Madagascar periwinkle. The structure of the linked vectors is as shown in FIG.
In FIG. 2, NPTII means the neomycin phosphotransferase II gene (kanamycin resistance gene), 35Spro means the cauliflower mosaic virus 35S promoter, and NOSPro means the noparin synthase gene promoter.

(4) Agrobacterium infection, sterilization treatment, and selection treatment
The above-mentioned Agrobacterium was inoculated into 2 ml of YEP liquid medium (bactobeptone 10 g / l, yeast extract 10 g / l, NaCl 5 g / l, pH 7.0) and cultured at 28 ° C. for 24 hours. The grown Agrobacterium was diluted about 100 times with sterile water, mixed well, and then the pre-cultured cotyledons were immersed for 10 minutes. The leaflets were taken out on a sterile filter paper, excess water was removed, and then the cells were transplanted to 1/2 B5 agar medium and cultured at 25 ° C. for 16 hours for 24 hours. The cells were transplanted into a 1/2 B5 medium containing 20 mg / l meropenem (Wako Pure Chemical Industries, Ltd.) or 300 mg / l cefotaxime (Wako Pure Chemical Industries, Ltd.) for eradication. Furthermore, one week later, the cells were transplanted into a 1/2 B5 medium containing 50 mg / l kanamycin (Nacalai Tesque, Inc.), 20 mg / l meropenem, or 300 mg / l cefotaxime, and selection was started. Selection was performed every 2 weeks on a medium having the same composition. Callus formation was observed about 2 weeks after infection with Agrobacterium, and rooting was observed from callus cultured in a medium containing meropenem about 1-2 weeks. Further continuation of culture of cotyledons with rooting resulted in shoot redifferentiation from callus. When the obtained shoots were transplanted to 1/2 B5 agar medium containing 100 mg / l kanamycin, rooting was confirmed. The rooted shoots were acclimated to soil and used as materials for genetic analysis.

(5) Gene analysis DNA was extracted from the leaves of redifferentiated plants acclimatized to soil using DNeasy Plant Mini Kit (Qiagen), and genomic PCR was performed using NPTII gene-specific primers (SEQ ID NOs: 18 and 19). gone. For the strains in which it was confirmed that the NPTII gene was introduced in the redifferentiated plant, RNA was extracted using RNeasy Plant Mini Kit (Qiagen) in order to confirm the expression of the further introduced gene, and Prome Script RT reagent Kit. (TAKARA Bio Inc.) was used to generate cDNA. RT-PCR analysis was performed using the introduced aroG4, trpE8, trpD, Nt-JAT2 gene-specific primers (SEQ ID NOs: 1-6, 14-15) to confirm the transcriptional expression of the introduced gene.

(6) Amino acid analysis 200 mg of leaf tissue was frozen in liquid nitrogen, disrupted using MM300, and then 500 μl of 80% ethanol was added. After shaking at room temperature for 30 minutes, the supernatant was transferred to a new tube by centrifugation at 12,000 rpm for 20 minutes at room temperature. 500 μl of 80% ethanol was added to the remaining precipitate, shaken at room temperature for 30 minutes, and then centrifuged at 12,000 rpm for 20 minutes at room temperature to transfer the supernatant to the previous tube. This work was repeated once again, and extraction was performed three times. The extract was adjusted to 1.5 ml, mixed well and 600 μl was transferred to another tube. These were dried using a vacuum evaporator, and 600 μl of sterile water was added and dissolved. Further, to remove the protein, 200 μl of chloroform was added, mixed well, and then centrifuged at 12,000 rpm for 10 minutes at room temperature, and the supernatant was transferred to another new tube. 20 μl of 0.2N hydrochloric acid was added to 180 μl of the purified solution, mixed well, and then filtered using an Ultra-free-MC 0.45 μm filter. The filtrate was subjected to amino acid analysis using a HITACHI high-speed amino acid analyzer L8800.

(7) Alkaloid analysis 200 mg of leaf tissue was frozen in liquid nitrogen, disrupted using MM300, and then 500 μl of 100% methanol was added. After shaking at 30 ° C. for 2 hours, the mixture was centrifuged at room temperature at 12,000 rpm for 20 minutes, and the supernatant was filtered using an Ultra-free-MC 0.45 μm filter, and the filtrate was used as a sample.
HPLC used a Capcelllpac C18 UG120 (4.6 x 250 mm, 5 μm, Shiseido) column with a flow rate of 1 ml / min using a mixture of 30% (v / v) methanol and 0.1 M phosphoric acid (pH 2). Deployed for 80 minutes. The temperature of the column was 40 ° C., and detection was performed by UV and fluorescence.

<< 2. Result >>
(1) Examination of Agrobacterium infection conditions (a) Selection of Agrobacterium Two types of Agrobacterium (Agrobacterium tumefaciens, LBA4404 (TAKARA Bio Inc.) and Agrobacterium including the pRI-201-AN binary vector) Infect the leaflets of Nichinichisou with one of Um tumefogenes, A13 (a strain deposited under the agricultural biological resource Genebank under registration number MAFF210266, a wild strain), containing 50 mg / l canamycin and 20 mg / l melopenem. As a result of selection and eradication with a medium, it was confirmed that no matter which Agrobacterium was infected with the leaflets, the formation of curls was confirmed, but no hairy roots or shoots were obtained with Agrobacterium tumefaciens. On the other hand, cotyledons infected with Agrobacterium risogenes were found to differentiate into hairy roots and shoots after the formation of callus (Table 2).
About 300 cotyledons were used for each Agrobacterium infection test. Since the test was conducted twice, a total of 600 cotyledons were used in each Agrobacterium infection test.

＊２０ｍｇ／ｌ メロペネム、５０ ｍｇ／ｌ カナマイシンを含む培地で選抜した場合。
＋：選抜可能、－：選抜不可能

* When selected in a medium containing 20 mg / l meropenem and 50 mg / l kanamycin.
+: Selectable,-: Not selectable

(B) Selection of disinfectant Next, Agrobacterium risogenes containing the pRI-201-AN binary vector, A13, and then infected with 300 mg / l cefotaxime or 20 mg / l meropenem. Um was sterilized and tested for shoot formation.
As a result of the test, no shoot differentiation was observed when cefotaxime was used.
On the other hand, it was found that meropenem can be used to form shoots.
In addition, in the medium containing 300 mg / l cefotaxime, the cotyledons were discolored, and then Agrobacterium growth was observed, and the sterilization effect was weak, whereas in the medium containing 20 mg / l meropenem, the growth of cotyledons was suppressed. It was also found that the suppression of Agrobacterium growth lasted for about 2 to 3 weeks (Table 3).
The reason why shoots are formed when meropenem is used is unknown, but when meropenem was used, unlike the case where cefotaxime was used, the growth of Agrobacterium was sufficiently suppressed, and the growth of Agrobacterium was sufficiently suppressed. Since it does not suppress the growth of cotyledons, it is speculated that the growth of Agrobacterium after the introduction of the gene into the plant was suppressed, and that the vigorous growth of the plant eventually led to the formation of shoots. rice field.

（a）＋：除菌良好、 －：アグロバクテリウムの増殖あり
（b）＋：子葉の生育良好、 －：子葉が黒く変色

(A) +: Good sterilization,-: Proliferation of Agrobacterium (b) +: Good growth of cotyledons,-: Black discoloration of cotyledons

(2) Preparation of transformant Based on the results of the above test, Agrobacterium risogenes, A13 was selected as the Agrobacterium used for transformation, and sterilized and selected with a medium containing 20 mg / l meropenem and 50 mg / l kanamycin. Was performed, and transplantation to a new medium was repeated every two weeks. As a result, about 4,200 leaf pieces were infected with Agrobacterium, and when selection and sterilization were repeated, 68 shoots were regenerated. When these shoots were grown in a medium containing 100 mg / l kanamycin, root formation was confirmed in 33 individuals (Table 4). After acclimatizing these strains, NPTII gene-specific primers were confirmed. When the genome PCR analysis was performed using, the introduction of the NPTII gene was confirmed in 24 strains, and the transformation rate was 0.6% (Fig. 2).

(3) RT-PCR analysis cDNAs were prepared from the leaves of the obtained 24 transformants, and the transcriptional expression of each introduced gene was analyzed by RT-PCR analysis. As a result, 5 individuals were obtained in which the expression of 3 genes of aroG4, trpE8, and trpD could be confirmed (Examples 1 to 5). In addition, 5 individuals were obtained in which gene expression of aroG4 or trpE8 and trpD was confirmed (Examples 6 to 10). Transformation analysis was performed on these.

＋：発現している、－:発現していない

+: Expressed,-: Not expressed

(4) Measurement of tryptophan content As a result of measuring the free tryptophan acid content using 5 lines of transformed leaves and non-transformed leaves (controls), the transformants were used as controls in all lines. A significant increase in tryptophan content was observed in comparison, and it was found that the tryptophan content was 3.6 to 6.5 times higher (Fig. 3).
In addition, the effects of the respective expressed genes were also shown in Examples 6 to 7 in which the expression of only the aroG4 gene was confirmed by RT-PCR analysis, and Examples 8 to 10 in which the expression of only trpE8 and trpD was confirmed. Amino acid analysis was performed to confirm. As a result, it was found that the tryptophan content also increased 4.2 to 6.2 times in these cases (Fig. 4).

(5) Measurement of alkaloid content As a result of measuring the alkaloid content using the transformed leaves and non-transformed leaves (controls) of Examples 1 to 5 obtained above, the transformed body was not found. It was found that the catharanthine content was 1.3 to 1.9 times higher than that of the transformant (control: control). It was also observed that some transformants tended to have high contents of bindolin and ajmalicine (Fig. 5). In addition, the effects of the respective expressed genes were also shown in Examples 6 to 7 in which the expression of only the aroG4 gene was confirmed by RT-PCR analysis, and Examples 8 to 10 in which the expression of only trpE8 and trpD was confirmed. Alkaloid analysis was performed to confirm. As a result, it was found that the catharanthine content was also increased in these strains (Fig. 6).
In addition, since catharanthine is located upstream of vinblastine and vincristine in the alkaloid synthesis pathway, it is considered that the content of vinblastine and vincristine also increases due to the increase in catharanthine content.
Similarly, transformants with increased ajmalicine content are considered to have increased serpentine content.

According to the present invention, it is possible to provide a transformed plant in which a foreign gene is integrated into the genome of a plant belonging to the genus Catharanthus of the Apocynaceae family, which has fertility. Further, according to the present invention, it is possible to provide a transformed plant capable of producing alkaloids with high efficiency. Therefore, the present invention is extremely useful industrially.

Claims (18)

A transformed plant in which the gene encoding the protein involved in tryptophan biosynthesis is integrated into the genome of a plant belonging to the genus Catharanthus in the family Apocynaceae as a foreign gene .
The gene encoding the protein involved in tryptophan biosynthesis is at least
(1) aroG or
(2) Combination of trpD and trpE
Including
However, the gene encoding the protein involved in tryptophan biosynthesis is conditioned on the fact that it does not contain the gene encoding ORCA3.
The transformed plant . The transformed plant according to claim 1, wherein the gene encoding the protein involved in the biosynthesis of tryptophan is stably expressed. The gene encoding the protein involved in the biosynthesis of tryptophan is a gene integrated into the genome of the plant using Agrobacterium rhizogenes and / or melopenem, according to claim 1 or 2. The transformed plant described. The transformed plant according to any one of claims 1 to 3, wherein the Agrobacterium retogenes is Agrobacterium rotogenes A13 strain (MAFF210266). The transformed plant according to any one of claims 1 to 4 , wherein the gene encoding the protein involved in the biosynthesis of tryptophan is a gene encoding the protein involved in the biosynthesis of tryptophan derived from Escherichia coli. .. Any of claims 1 to 5, wherein the gene encoding the protein involved in tryptophan biosynthesis is linked to a DNA sequence encoding a signal for translocating aldolase derived from a plant of the genus Catharanthus of the Apocynaceae family to chloroplast. The transformed plant according to item 1. Claimed that the synthetic amount of at least one alkaloid per unit mass of the transformed plant is higher than the synthetic amount of at least one alkaloid per unit mass of a wild plant strain of the genus Catharanthus in the family Apocynaceae. The transformed plant according to any one of 1 to 6. The synthetic amount of at least one alkaloid per unit mass of the transformed plant is 1.1 times or more higher than the synthetic amount of at least one alkaloid per unit mass of the wild strain of the plant of the genus Catharanthus in the family Apocynaceae. The transformed plant according to any one of claims 1 to 7 . The transformed plant according to claim 7 or 8, wherein the alkaloid is at least one selected from the group consisting of catharanthine, bindrin, ajmalicine, vinblastine, vincristine, tabersonine, serpentine, stenmadenin, and strictocidin. The transformed plant according to any one of claims 7 to 9, wherein the alkaloid is at least one selected from the group consisting of catharanthine, bindrin, and ajmalicine. The transformed plant according to any one of claims 7 to 10 , wherein the alkaloid is catharanthine. The transformed plant according to any one of claims 1 to 11 , wherein the gene encoding the protein involved in the biosynthesis of tryptophan comprises aroG, trpD and trpE. The aroG is a gene modified to suppress the feedback control of 3-deoxy-D-arabino-hepturosonic acid-7-phosphate synthase by phenylalanine and / or.
The transformed plant according to any one of claims 1 to 12 , wherein trpE is a gene modified so as to suppress feedback control of anthranilic acid synthase by tryptophan. The aroG is a gene having the DNA sequence of SEQ ID NO: 21 and / or the trpD is a gene having the DNA sequence of SEQ ID NO: 22 and / or the trpE has the DNA sequence of SEQ ID NO: 24. The transformed plant according to any one of claims 1 to 13 , which is a gene. The transformed plant according to any one of claims 1 to 14, wherein the plant belonging to the genus Catharanthus of the Apocynaceae family is Madagascar periwinkle. A plant piece obtained from the transformed plant according to any one of claims 1 to 15. A step of introducing a gene encoding a protein involved in tryptophan biosynthesis into a plant belonging to the genus Catharanthus in the family Apocynaceae as a foreign gene using Agrobacterium rhizogenes .
If the plant of the genus Catharanthus in the family Apocynaceae into which a gene encoding a protein involved in tryptophan biosynthesis is not a callus, a step of forming a callus from the plant into which the gene has been introduced, and
The method for producing a transformed plant according to any one of claims 1 to 15, which comprises a step of differentiating roots and shoots from callus using a medium containing melopenem to obtain a transformed plant. A step of introducing a gene encoding a protein involved in tryptophan biosynthesis into a plant belonging to the genus Catharanthus of the Apocynaceae family using Agrobacterium rhizogenes .
If the plant of the genus Catharanthus in the family Apocynaceae into which a gene encoding a protein involved in tryptophan biosynthesis is not a callus, a step of forming a callus from the plant into which the gene has been introduced, and
A method for producing an alkaloid, which comprises a step of differentiating roots and shoots from callus using a medium containing melopenem to obtain a transformed plant.

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