KR100973997B1 - Recombinant vector comprising polynucleotide encoding isoflavone synthase and transformants transformed thereby - Google Patents

Abstract

The present invention relates to a recombinant expression vector comprising a polynucleotide encoding an isoflavone synthase and a transformant using the same, and more particularly, to a promoter and an isoflavone synthetase of SEQ ID NO: 3 operably linked thereto. A recombinant expression vector comprising a polynucleotide and a transformant using the same. The recombinant vector of the present invention enables the synthesis of isoflavones by stably expressing isoflavone synthase in rice that does not synthesize isoflavones, thereby producing a transformant containing a higher concentration of isoflavones than a conventional transformant. It has the effect of making it possible. Therefore, the recombinant vector of the present invention can be used for the purpose of the development of a new breed of transgenic crops that biosynthesize the useful material isoflavones.

Isoflavones, transformants, isoflavone synthase, introns

Description

Recombinant vector comprising polynucleotide encoding isoflavone synthase and transformants transformed thereby

The present invention relates to a recombinant expression vector comprising a polynucleotide encoding an isoflavone synthase and a transformant using the same, and more particularly, to a promoter and an isoflavone synthetase of SEQ ID NO: 3 operably linked thereto. A recombinant expression vector comprising a polynucleotide and a transformant using the same.

As molecular biology techniques have evolved, methods have been developed to generate useful metabolites by genetic engineering methods or to change them into more useful substances. Among them, plant metabolic engineering using plants as a medium or plant molecular farming researches that produce non-plant-derived high value oral vaccines and medical proteins in plants Although much has been achieved, due to various technical difficulties, the specific results are not so many.

One such difficulty is that the expression of the transformed genes is often poor. Inhibition of such expression is caused by a variety of causes, such as inhibition of expression at the gene level, degradation of the expressed protein, loss of function of the expressed protein, and the cause of each transformation is not precisely identified. It is difficult to provide a solution.

Moreover, IFS1 and IFS2 derived from soybeans Genes belong to the cytochrome P450 family, and the proteins encoded by these genes are known to be very unstable. Therefore, the production of target isoflavones has been reported to be very low in the examples that have been transformed using these genes that do not include introns, ie, Arabidopsis, corn BMS (Black Mexican Sweer) cell lines, and tobacco plants. (Yu et al., Plant Physiology 124: 781-793, 2000). Moreover, plant cytochrome P450s are known to be very difficult to isolate from plants due to their scarcity, many similar proteins, instability, etc. (Akashi et al., Plant Physiology 121: 821-828, 1999). Therefore, in order to obtain target traits through transformation using these genes, access through separation of proteins is difficult, and stable expression of these genes in cells is essential.

On the other hand, isoflavone is a representative physiologically active substance of soybean and is produced only in soybean crops including soybeans. In addition to its effects on breast cancer, heart disease, and prostate cancer, it is known to act as a powerful antioxidant, such as eliminating blood clots (Jung et al., Nat. Biotech. 18: 208-212, 2000).

Therefore, the present inventors have studied to develop a transgenic plant that synthesizes isoflavones. When using the IFS genes of SEQ ID NO: 1 and SEQ ID NO: 2 isolated from soybeans, the present inventors can prepare a transformant to synthesize isoflavones at a high concentration. It was confirmed that the present invention was completed.

Accordingly, an object of the present invention is to provide a recombinant vector for plant transformation capable of biosynthesizing isoflavones using the IFS2 gene including an intron and a transformant using the same.

In order to achieve the above object, the present invention provides a method for producing a transformed rice using a recombinant vector for plant transformation comprising the IFS2 gene.

In order to achieve another object of the present invention, there is provided an isoflavone production method characterized in that the isoflavones are extracted from the transformed rice using a recombinant vector for plant transformation comprising the IFS2 gene.

Hereinafter, the content of the present invention will be described in more detail.

The recombinant vector of the present invention is characterized in that it comprises a promoter and a polynucleotide of SEQ ID NO: 3 operably linked thereto.

Preferably the polynucleotide of the present invention may be DNA or RNA consisting of the nucleotide sequence represented by SEQ ID NO: 3. In this case, when the polynucleotide of the present invention is RNA, thymine (T) of SEQ ID NO: 3 may be understood to be replaced by uracil (U). The polynucleotide can be prepared by known chemical synthesis.

SEQ ID NO: 3 is an IFS2 gene as an isoflavone synthase gene isolated from soybean. Isoflavone synthase genes isolated from soybeans have two isoforms, IFS1 and IFS2. IFS1 genes are constantly expressed in soybean developmental stages, while IFS2 genes have the highest isoflavone content. The expression level increases with the developmental stage (Dhaubhadel Plant Physiol. 143: 326-338). This is thought to reflect the difference in isoflavone synthesis efficiency of the two isotopes. In vitro , the isoflavone synthesis efficiency of the two isotopes using the yeast expression system showed that the IFS1 is twice as high as the isoflavone compared to the IFS2. It is known to synthesize (Jung et al., Nat. Biotech. 18: 208-212, 2000).

Isoflavones are produced from the phenylpropanoid pathway, where most of the plant's secondary metabolites are made, and from the intermediate substrates of the phenylpropanoid pathway, naringenin and liquiritigenin. Under the action of isoflavone synthase, isoflavones genistein and daidzein are produced respectively (Yu et al., Plant Physiology 124: 781-793, 2000). Isoflavone action of ileonaneunde synthase is a two-step process, non-2S- Plastic bar (2S-flavanone) are isometric receive the action of the flavone synthase 2-hydroxy-iso-flavanone (2-hydroxyisoflavanone) is then either voluntarily or plant Isoflavones are produced by specific dehydratases in the body (Yu et al., Plant Physiology 124: 781-793, 2000).

The inventors have expressed the enzymes using recombinant vectors comprising a promoter and a polynucleotide encoding the IFS2 gene operably linked thereto, thereby making it possible to synthesize isoflavones in rice.

The recombinant vector of the present invention can be prepared using a known plant transformation vector as a base vector, and a general binary vector or a cointegration vector can be used. A wide variety of binary vectors are widely used in plant transformation, and almost all binary vectors are international centers and university research institutes, such as the Center for the Application of Molecular Biology to International Agriculture, GPO Box 3200, Canberra ACT2601, Australia (CAMBIA). The basic binary vector skeleton can be used by variously modifying a transformant selection marker gene, a promoter, a transcription termination gene, and the like at the left and right boundary sites where the gene is delivered using the Ti plasmid as a parent.

Since the vector according to the present invention is a plant transformation vector, various plant-functional promoters known in the art may be used, and for the expression of the polynucleotide of SEQ ID NO: 3, the polynucleotide is operably linked with the promoter. The term 'promoter' refers to a DNA sequence that regulates the expression of a nucleic acid sequence operably linked in a particular host cell. 'Operably linked' means that one nucleic acid fragment is combined with another nucleic acid fragment. Its function or expression is affected by other nucleic acid fragments. In addition, it may further comprise any operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosomal binding site, and a sequence regulating termination of transcription and translation.

The promoters include the cytochrome c gene-derived OsCc1 promoter, rice globulin (Glb) promoter, cauliflower mosaic virus (CaMV) 35S promoter, pigwarm mosaic virus 35S promoter, and sugarcane basil that are known to be expressed in rice seeds. Reform Virus Promoter, Comerina Yellow Mottle Virus Promoter, Light-Induced Promoter from Subunit of Ribulose-1,5-bis-phosphate Carboxylase, ssRUBISCO, Rice Cytozolic Triophosphate Isomerase (TPI) Promoter, adenine phosphoribosyltransferase (APRT) promoter from Arabidopsis, a rice actin 1 gene promoter and a mannopin synthase and octopin synthase promoter, preferably an OsCc1 promoter derived from cytochrome c gene ( Jang et al., Plant Physiol. 129: 1473-1481, 2002) It is: (113-125 Qu & Takaiwa, Plant Biotech J. 2.) Type specific expression glow Evelyn (globulin, Glb) promoter.

In addition, the selection marker genes include antibiotic resistance genes, herbicide resistance genes, metabolic genes, luminescent genes, GFP (green fluorescence protein) genes, GUS (β-glucuronidase) genes, GAL (β-galactosidase) genes, etc. It is not limited. Specifically herbicide resistant bar Gene, neomycin phosphotransferase II (NPT II) gene, hygromycin phosphotransferase gene, phosphinothricin acetyltransferase gene, chloramphenicol acetyltransferase or dihydrofolate reductase gene, etc. Herbicide resistant bar genes are preferred.

Preferably, the vector for plant transformation of the present invention may be a vector having a cleavage map disclosed in FIG. 1, ie, a pMJ-GB- IFS2 or a pMJC-GGB- IFS2 vector. The vector is a pSB11 vector (Genbank Accession No. AB027256) as a backbone of the cytochrome c gene-derived OsCc1 promoter or rice endosperm globulin ( Glb ) promoter and potato protease inhibitor (Protease inhibitor II, PinII ) AttB1 sequence between terminators of; The IFS2 gene of SEQ ID NO: 3; And attB2 sequences in sequence.

Standard recombinant DNA and molecular cloning techniques, molecular biology techniques known in the art, are described in Sambrook, J., Fritsch, EF and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd ed. , Cold Spring Harbor Laboratory: Cold Spring Harbor, NY, 1989; by Silhavy, TJ, Bennan, ML and Enquist, LW, Experiments with Gene Fusions, Cold Spring Harbor Laboratory: Cold Spring Harbor, NY, 1984; and by Ausubel, FM et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-lnterscience, 1987).

Meanwhile, in one embodiment of the present invention, the recombinant vector of the present invention is transformed into Agrobacterium agrobacterium tumefaciens LBA4404 , and then the gene of the present invention is introduced into plant cells by the transformed Agrobacterium. It was.

Thus, the present invention provides cells transformed with the recombinant vector of the present invention. The transformed cells include Agrobacterium spp. And Escherichia. coli), Bacillus subtilis (Bacillus subtilis), Streptomyces (Streptomyces), Pseudomonas (Pseudomonas), Proteus Mira Billy's (Proteus prokaryotic host cells such as mirabilis ) or Staphylococcus , fungi (eg Aspergillus ), yeast (eg Pichia Pastries ( Pichia pastoris), saccharide as MY processes Sergio non jiae (Saccharomyces cerevisiae), investigating car break in Rome Seth (Schizosaccharomyces), Castello La Neuro Chrysler Corporation (Neurospora It may be a cell derived from higher eukaryotes, including lower eukaryotic cells such as crassa ), insect cells, plant cells, mammals, etc., but is not limited thereto, and preferably Agrobacterium tumefaciens ( Agrobacterium) tumefacience ) or Agrobacterium lyzogenes rhizogenes ).

The present invention also provides a transgenic plant cell into which the vector is introduced and synthesizes isoflavones.

The preparation of the transformed plant cells may use transformation methods known in the art for introducing vectors into plants. For example, but not limited to, Agrobacterium spp.) Microbial transformation, particle gun bombardment, Silicon carbide whiskers, sonication, electroporation and precipitation by PEG (Polyethylenglycol) Can be used. Preferably, Agrobacterium-mediated transformation can be used, and the methods exemplified in the literature can be used (Horsch et al., Science 227: 1229-1231, 1985). For example, Agrobacterium-mediated transformation methods for rice are known in the art and the like (An et al., EMBO J., 4: 227-288,1985).

The plant cells may be cultured according to a conventional method to be liquid cultures, callus, plasma cultures, and may be differentiated to be plant tissues or plants. That is, the transformed cells into which the recombinant vector of the present invention is introduced can be re-differentiated into plants through processes such as callus induction, rooting, and soil purification using standard techniques known in the art, and asexual, such as folding and grafting. It can be produced by the planetary breeding method using the breeding method and the seed.

Cultivation of plant cells may be carried out by any method known to those skilled in the art, such as separating a part of a plant from a mother and cultivating it aseptically under suitable conditions, such as liquid culture of tissue sections, callus culture of tissue sections, and protoplast culture. The culture conditions and methods may be carried out by conditions and methods known to those skilled in the art.

Differentiating the cultured plant cells into plants is to induce differentiation of cultured plant cells of callus, protoplast form under appropriate conditions to differentiate into plant tissues or plants. It may be carried out by the method.

Accordingly, the present invention also provides a transgenic plant into which the vector is introduced and synthesizes isoflavones.

At this time, the plant as used herein includes the whole plant (whole plant), a part of the plant, callus, plant tissue, plant cells and plant seeds. Plants to which the method according to the present invention may be applied include monocotyledonous plants or dicotyledonous plants. Examples of the monocotyledonous plant include, but are not limited to, rice, wheat, barley, bamboo shoots, corn, taro, asparagus, onions, garlic, leeks, leeks, soothing, hemp and ginger. Examples of dicotyledonous plants include, but are not limited to, baby pole, eggplant, tobacco, pepper, tomato, burdock, garland chrysanthemum, lettuce, bellflower, spinach, beetroot, sweet potato, celery, carrot, buttercup, parsley, cabbage, cabbage, mustard, There are watermelons, melons, cucumbers, pumpkins, gourds, strawberries, soybeans, green beans, kidney beans, buzzfoot trefoils, potatoes, duckweed, perilla, pigeon beans, and peas.

In exemplary embodiments, in order to remove the genomic DNA of the IFS1 and IFS2 gene used in this invention to remove the genomic DNA from sinpal dalkong second arc leaves are known to a high isoflavone content and, IFS1 and IFS2 gene by the PCR method of the present invention Was cloned.

In another embodiment of the present invention, the cloned IFS1 and IFS2 genes are introduced into the pSB11 vector, thereby providing pMJC-GB- IFS1, pMJC-GB- IFS2 , pMJC-GGB- IFS1. And pMJC-GGB- IFS2 were prepared and introduced into the Agrobacterium LBA4404 strain.

In another embodiment of the present invention using the transformed Agrobacterium strain was transformed rice to prepare a transformed rice to synthesize isoflavones.

In another embodiment of the present invention it was confirmed whether the gene introduced in the transformed rice of the present invention, whether expression and synthesis of isoflavones. As a result, it was found that IFS1 and IFS2 genes were introduced and expressed in all of the transgenic rice, and in the transgenic rice using OsCc1 promoter for gene expression, there was no synthesis of Zenithine , a kind of isoflavone, but Glb promoter In the transgenic rice using the degree of difference, but the synthesis of Zenithine was found to be well made. In particular, the content of isoflavones was slightly different depending on the individual transformed, but IFS2 Genetically Modified Rice IFS1 It was found that the amount of isoflavones synthesized was higher than that of rice transformed with the gene. This is IFS2 It is considered that the activity of IFS2 expressed by the gene is stronger.

Accordingly, the present invention provides a recombinant vector for isoflavone synthesis. The recombinant vector of the present invention enables the synthesis of isoflavones by stably expressing isoflavone synthase in rice that does not synthesize isoflavones, thereby producing a transformant containing a higher concentration of isoflavones than a conventional transformant. To help.

Below. The present invention will be described in detail by way of examples.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

< Example  1>

IFS1 and IFS2  Gene Cloning

In order to isolate genomic DNAs of IFS1 and IFS2 genes used in the present invention, genomic DNA was extracted from leaves of Sinpalludo No. 2 known to have high isoflavone content (Rogers & Benich, In: Gelvin SB, Schilperoort RA, Verma DPS ( eds) Plant Molecular Biology Manual.Kluwer Academic Publishers, Dordrecht, The Netherlands, pp 1-8, 1994). Using 10 ng of isolated genomic DNA as a template, 25 pM of each primer and 50 μl of final volume of PCR reaction solution (1 × f-Taq buffer (Solgent, Korea), 50 μM of each dNTP, 1 × band doctor (Solgent, Korea), PCR was performed using f-Taq DNA polymerase 1.5 units (Solgent, Korea) and residual secondary distilled water). At this time, as a primer for cloning IFS1 , a combination of SEQ ID NO: 5 (IFS1FOR: 5'-GTAATTAACCTCACTCAAACTCGG-3 ') and SEQ ID NO: 6 (IFS2 REV: 5'-GCAAACGAAGACAAATGGGAGATGATA-3') was used, and the IFS2 gene was used. As a primer for cloning, a combination of SEQ ID NO: 7 (IFS2FOR: AAAATT AGCCTCACAAAAGCAAAG-3 ') and SEQ ID NO: 8 (IFS2REV: 5'-GCAAACGAAGAC AAATGGGAGATGATA-3') was used. PCR conditions were 94 ° C. 4 minutes; 30 cycles of 94 ° C. 15 seconds, 55 ° C. 30 seconds, 72 ° C. 60 seconds; And 72 ° C. for 7 minutes.

The amplified PCR product was subjected to electrophoresis on 0.8% agarose gel to which EtBr (ethidium bromide) was added, and then recovered from the gel and cloned into pGEM-T easy vector (Promega, USA) for sequencing. .

As a result, an IFS1 gene having a nucleotide sequence of SEQ ID NO: 1 and an amino acid sequence of SEQ ID NO: 2 and an IFS2 gene having a nucleotide sequence of SEQ ID NO: 3 and an amino acid sequence of SEQ ID NO: 4 were obtained.

As a result of comparing and analyzing the cloned IFS1 gene and IFS2 gene with the sequence previously registered in NCBI, the nucleotide sequence and amino acid sequence of the cloned IFS1 gene in the present invention was previously registered in NCBI (Nucleotide NCBI Accession No .: AF195798 Amino acid NCBI Accession No .: AAF45142), and the nucleotide sequence and amino acid sequence of the IFS2 gene were previously registered in NCBI (Nucleotide NCBI Accession No .: AF195819; Amino acid NCBI Accession No .: AAF45143). The homology was 98.7% and 98.1%, respectively.

< Example  2>

Construction of Carrier for Transformation and Introduction to Agrobacterium

IFS1 and IFS2 genes, including the introns used for the present invention, were constructed using pSB11 vectors (Komari et al. ) Using a gateway system (Invitrogen, USA). al ., Plant J. 10: 165-174, 1996). IFS1 and IFS2 For gene expression, seed-specific Glb promoters isolated from rice and OsCc1 promoters derived from cytochrome c genes known to be expressed in rice seeds were used.

Transfection vehicle for switching the introduction of the genes identified are not under control by the promoter OsCc1 pMJC-GB- and IFS1 pMJC-GB- IFS2, it is under the control Glb promoter was named pMJC-GGB- IFS1 and pMJC-GGB- IFS2. Bar controlled by the CaMV 35S promoter for selection of transformed callus and plants (Bialaphos resistance) gene was used, and a matrix attachment region (MAR) was added inside both borders to induce stable expression of the gene. The major gene sequence of the recombinant plant transformation carrier is shown in FIG. 1.

The transformed binary vectors were introduced into the Agrobacterium LBA4404 strain by tri-parental mating, and two antibiotics, spectinomycin, were selected for selection of Agrobacterium containing the recombinant plasmid. (spectinomycin) and tetracycline (tetracycline) were used. Transformation into Agrobacterium was confirmed by colony PCR. PCR reactions were performed with 1X f-Taq buffer (Solgent, Daejeon, Korea), 50 μM of each dNTP, 1X band doctor (Solgent, Daejeon, Korea), 1.5 units of f-Taq DNA polymerase (Solgent, Daejeon, Korea) and each 25 pM of primer was added and a portion of the colonies transformed with each carrier was added to the PCR reaction solution in which the final volume was adjusted to 25 μl with sterile water. PCR 94 min 4 min; 30 cycles of 94 ° C 15 seconds, 55 ° C 30 seconds, 72 ° C 60 seconds; And 72 ° C. for 7 minutes. The amplified product was confirmed by electrophoresis on 0.8% agarose gel.

As a result, as shown in FIG. 2, the bands were displayed at about 1.7 kb positions, which is the expected size of the primers used in all 10 selected colonies (1 to 10), indicating that the IFS1 and IFS2 genes were stably transformed into Agrobacterium. Confirmed. This transformed Agrobacterium was used for the transformation of rice after propagation.

< Example  3>

Isoflavones  Preparation of Transgenic Rice to Synthesize

<3-1> Callus  Judo and starters

The varieties used were Nakdong rice, a beautiful rice, and Black Namb rice, a colored rice. First, the brown rice with the seed shells removed was washed for 5 minutes in 70% ethanol and then sterilized twice at room temperature for 15 minutes with 50% lacx solution and then sterilized by washing at least 10 times with sterile water. The sterilized seeds were inoculated with about 15 to 20 (90 × 20 cm petridish) in 2N6 medium containing 2,4-D 2 mg / L (see Table 1) with the belly up, followed by 3 Callus was induced by incubation for −4 weeks (FIG. 3A). Among the callus induced callus was selected in a good condition without the change of a constant round shape of 1-2 mm was grown in 2N6 medium for 3 days and used for transformation.

<3-2> Agrobacterium culture and suspension preparation

Each Agrobacterium transformed with four recombinant plasmids was streaked streaked in AB-ST medium (see Table 1) and incubated for 4 to 5 days at 28 ° C dark conditions. The cultured Agrobacterium was dispensed with 15-20 ml AAM medium (see Table 1) per petri dish, scraped off using a sterile loop and transferred to a falcon tube. It was then vortaxed for several seconds so that Agrobacterium could be well suspended, and the final inoculation concentration was 1.5-2.0 at OD650 nm.

<3-3> Agrobacterium inoculation

Callus grown for 3 days was transferred to an empty Petri dish, and then inoculated with about 20 to 30 minutes by pouring 15-20 ml of Agrobacterium suspension. After inoculation, the callus was transferred to a petri dish containing sterilized filter paper to remove the remaining Agrobacterium suspension, which was then wound into a co-culture medium.

Type and composition of medium for each stage of transformation  Transformation stage Badge Furtherance Callus induction
And proliferation 2N6 N6 macro, micro salts, N6 vitamins. Casamino acid 300mg / L, proline 500mg / L, sucrose 30g / L, glutamine 500mg / L, gelite 2.5g / L, 2.4-D 2mg / L, pH 5.8 Agrobacterium culture AB-ST AB buffer, salt, glucose 5 g / L, Bacto-agar 15 g / L, spectinomycin 50 mg / L, tetratracycline 10 mg / L, pH 7.0 Agrobacterium Suspension and Inoculation AAM AA macro, micro salt, amino acid stock, MS vitamin, iron stock, casamino acid 500 mg / L, sucrose 68.5 g / L, glucose 36 g / L, acetosyringone 100 uM / L, pH 5.8 Co-culture 2N6-AS 2N6 medium, acetosyringone 100uM / L, glucose 10g / L, gelite 2.5g / L, 2.4-D 2mg / L, pH 5.2 1 / 2-2N6- AS-New 1 / 2-2N6 medium, acetosyringone 100uM / L, glucose 10g / L, L-cysteine 10.5mg / L, sodium thiosulfate 1mM, dithiothreitol 1mM, silver nitrate 5mg / L, gelite 3.0g / L, 2.4 -D 2 mg / L, pH 5.2 Transformation
Callus starter 2N6-CP 2N6 medium, 250 mg / L cefotaxime, 6 mg / L L-phosphinotricine, 2.5 g / L gelite, 2.4-D 2 mg / L, pH 5.8 subdivision MSR-CP MS macro, micro salts, MS vitamins, Cytotaxin 250 mg / L, NAA 1 mg / L, Kinetine 5 mg / L, Sorbitol 30 g / L, Maltose 20 g / L, L-phosphinothricin 3 mg / L, Zeolite 4 g / L, pH 5.8 Root induction MS0 MS macro and micro salts, MS vitamin, sucrose 30 g / L, gelite 2.5 g / L, pH 5.8

Components and Additions of Callus Induced and Plant Regeneration Media Callus induction subdivision N6 (Chu) Badge Amount MS badge Amount N6 macro salt
KNO ₃
(NH ₄ ) ₂ SO ₄
KH ₂ PO ₄
MgSO ₄
(Or MgSO ₄ 7H ₂ O)
CaCl ₂
(Or CaCl ₂ 2H ₂ O)
2.83g / l
463 mg
400 mg
90 mg
(Or 185 mg)
125.33 mg
(Or 166 mg) Macro salt
KNO ₃
NH ₄ NO ₃
KH ₂ PO ₄
MgSO ₄
CaCl ₂
1900 mg
1650 mg
170 mg
180.54 mg
332.02 mg iron
(Or FeNaEDTA)
36.70mg iron
FeNaEDTA
36.70 mg N6 Micro Salts
MnSO ₄ H ₂ O
ZnSO ₄ 7H ₂ O
H ₃ BO ₃
KI

Na ₂ MoO ₄ 2H ₂
CuSO ₄ 5H ₂ O
CoCl ₂ .6H ₂ O
3.3mg
1.5mg
1.6mg
0.8mg

-
-
- Fine salt
MnSO ₄ H ₂ O
ZnSO ₄ 7H ₂ O
H ₃ BO ₃
KI

Na ₂ MoO ₄ 2H ₂
CuSO ₄ 5H ₂ O
(Or CuSO ₄ )
CoCl ₂ .6H ₂ O
16.9 mg
8.6 mg
6.20mg
0.83 mg

0.25 mg
0.025mg
Or 0.016 mg
0.025mg N6 Vitamins
Nicotinic acid
Thiamine HCl
Pyridoxine HCl
Glycine

0.5mg
0.1mg
0.5mg
2.0mg
vitamin
Nicotinic acid
Thiamine HCl
Pyridoxine HCl
Glycine
Myo-inositol
0.5mg
0.1mg
0.5mg
2.0mg
100mg

<3-4> Co-culture

The medium used for the co-culture with Agrobacterium was reduced to 1/2 the content of the co-culture medium composition base medium (2N6) in order to improve the introduction of the target gene into the plant cells and increase the survival rate of the transformed cells. In order to minimize the oxidative destruction of plant cells generated during the coculture process, three antioxidant compounds, namely, 10.5 mg / L L-cysteine, 1 mM sodium thiosulfate, and 1 mM dithiothritol, were added. Agrobacterium To inhibit excessive growth and ethylene activity, 5 mg / L of silver nitrate was added, and a medium containing 3.0 g / L of the amount of added gelite was used to prevent the regression of the medium due to the addition of silver nitrate. .

Inoculated callus increased the stability of the inoculated cells by covering one sheet of filter paper (Watman # 1) on the co-culture medium and the temperature during the co-cultivation period was changed to about 3 ℃. The co-culture period was 5 days and no damage from overgrowth of Agrobacterium was observed with this method.

<3-5> Selection of Transformed Callus

After the coculture process, callus was washed at least 10 times with a solution containing 250 mg / L of cefotaxim to remove Agrobacterium, and the washed callus was transferred to a Petri dish containing sterile filter paper. The remaining solution was completely removed. After this process, the callus was transferred to 2N6-CP medium containing PPT (L-phosphinotricine (6 mg / L) (see Table 1)) and cultured for 3 weeks at 27 ° C dark conditions to select transformed callus as the primary. The selected callus was secondarily selected by culturing for 2 weeks under the same conditions in the same medium (FIG. 3B).

<3-6> Induction of Regenerated Plants from Transformed Callus

Selected callus was 3 mg / L PPT, 250 mg / L Cytotaxin, 1 mg / L Naphthalene Acetic Acid (NAA), 5 mg / L Kinine (Kinetine), 30 g / L Sorbitol and 20 g / L Maltose Transgenic plants were induced by incubation in -CP medium (see Table 1) and then cultured in 25 ° C bright conditions (Fig. 3C).

<3-7> Root Induction and Purification

Replanted plants were transferred to MS0 medium without growth hormone (see Table 1) to induce roots. The root-derived plants were purified by immersion in a 1000-fold hypoxic aqueous solution for about 7 to 10 days (FIG. 3D), and the purified plants were transplanted into pots and managed in a greenhouse (FIG. 3E of FIG. 3). H).

<Example 4>

Gene introduction and expression analysis of transgenic rice

<4-1> Confirmation of introduction of gene into transformant

In order to confirm the introduction of the transformant gene was confirmed whether the introduction of the gene at the DNA level using the PCR as follows. DNA was isolated from the rice leaves of each transformant (TG1 to TG10) by CTAB method.

The isolated DNA was used as a template, and for the IFS1 gene, a combination of SEQ ID NO: 5 (IFS1FOR: 5'-GTAATTAACCTCACTCAAACTCGG-3 ') and SEQ ID NO: 6 (IFS2 REV: 5'-G CAAACGAAGACAAATGGGAGATGATA-3') was used as a primer. For the IFS2 gene, PCR was performed using a combination of SEQ ID NO: 7 (IFS2FOR: AAAATTAGCCTCACAAAA GCAAAG-3 ') and SEQ ID NO: 8 (IFS2REV: 5'-GCAAACGAAGACAAATGGGAGATG ATA-3') as a primer. PCR 94 min 4 min; 30 cycles of 94 ° C. 15 seconds, 55 ° C. 30 seconds, 72 ° C. 60 seconds; And 72 ° C. for 7 minutes. The amplified PCR product was confirmed by electrophoresis on agarose gel.

As a result, as shown in Fig. 4A, PCR products were confirmed in all the transformants to confirm that IFS1 and IFS2 genes were introduced, respectively (see Fig. 4A).

<4-2> Confirmation of gene expression in transformants

RT-PCR was performed to investigate the expression levels of IFS1 and IFS2 genes in each transformant. Total RNA was isolated from the rice leaves of each transformant (TG1 to TG10) using Trizol (Sigma-Aldrich, USA) following the manufacturer's method.

Using the ProSTAR ^TM First-Strand RT-PCR system (Stratagene, USA) from the isolated whole RNA, cDNA was synthesized according to the manufacturer's method, which was used as a template, and IFS1. For the gene, a combination of SEQ ID NO: 5 (IFS1FOR: 5'-GTAATTAACCTCACTC AAACTCGG-3 ') and SEQ ID NO: 6 (IFS2REV: 5'-GCAAACGAAGACAAATGGGAGA TGATA-3') was used as a primer, and for IFS2 gene, SEQ ID NO: 7 (IFS2FOR : PCR was performed using a combination of AAAATTAGCCTCACAAAA GCAAAG-3 ') and SEQ ID NO: 8 (IFS2REV: 5'-GCAAACGAAGACAAATGGGAGATG ATA-3') as a primer. PCR 94 min 4 min; 30 cycles of 94 ° C. 15 seconds, 55 ° C. 30 seconds, 72 ° C. 60 seconds; And 72 ° C. for 7 minutes. The amplified PCR product was confirmed by electrophoresis on agarose gel. The amplified PCR product was confirmed by electrophoresis on agarose gel.

As a result, as shown in Figure 4B, IFS1 and IFS2 in both transformants It was confirmed that the gene is expressed (see FIG. 4B).

<4-3> of the gene introduced into the transformant Copy number  Confirm

To identify the copy number of each gene introduced into the transformants, the control plants (Black and White Rice) and the transformants ( OsCc1 promoter and Southern blot was performed as follows using rice transformed with the gene under the control of the Glb promoter.

Genomic DNA was isolated from the leaves of control plants and transformants as in Example <4-1>. For the 10 ug transgenic genomic DNA under the control of the promoter OsCc1 body with Bam HI, Glb promoter under the control transformant it was restricted enzymatic treatment with Dra I. It was then electrophoresed overnight in a 0.8% gel and then transferred to a nylon membrane. As probes, IFS1 and IFS2 genes labeled with ³² P were used.

As a result, as shown in Figure 4C, it was found that the IFS1 and IFS2 genes were mainly introduced in 1 to 3 copies in the transformant.

< Example  5>

In transformed rice Isoflavone  Synthetic Check

Synthesis of Zenithine, a kind of isoflavones, and its amount in the transformed rice were confirmed as follows. Rice seeds were ground with liquid nitrogen and then extracted with 80% methanol for isoflavone content analysis from rice seeds. This was filtered through an injection filter (Acrodisc CR-PTFE injection filter), and then 3 ml of 1N HCl was added to 1 ml of extract to hydrolyze the glycoside form of isoflavones and incubated at 95 ° C for 2 hours. And it was extracted with 1 ml of ethyl acetate.

The extract was analyzed by high-performance liquid chromatography (HPLC), and HPLC analysis conditions were performed using XTerra MS C18, 5 μm (150 × 2.1 mm), and 2% acetic acid solution (2% acetic acid) as solvent. / in water, eluent A) and 0.5% acetic acid / acetonitrile mixture (0.5% acetic acid / in water and acetonitrile (50:50, v / v), eluent B) and flow rate of 0.2 ml / min The spectrum was measured at 450 nm wavelength at UV 200. 2 μl of the sample was injected. Zenithstein peaks were identified based on the UV spectrum of the standard sample and the remaining time. The content of isoflavones (zenithstein) is obtained by injecting standard reagents from Sigma, USA, and injecting into HPLC to obtain the area of the standard material, and then extracting the sample extract. g) (Kim et al., J. Agric. Food Chem. 54: 10003-10010, 2006).

In addition, HPLC / MS analysis was performed to confirm the molecular weight of the biosynthesized isoflavone Zenithine. ZMD 4000 (Micromass, UK) was used as an analyzer and nitrogen was used as a spraying gas. The source temperature was 150 ° C., the decomposition temperature was 250 ° C., and the amplification voltage was 650 V to monitor ions having a 270.9 m / z value corresponding to Zenithsteine. Zenithstein was identified on the basis of the UV spectra, retention time and m / z values of the standard samples.

As a result, as shown in Figure 5, in the case of black rice paddy control isoflavones such as Zenithine was not synthesized (Fig. 5A). In addition, under OsCc1 promoter control Isoflavones such as Zenithine were not detected in the TG 9-1 line, which is a rice transformant expressing IFS1 (FIG. 5B). However, isoflavone zenithine was detected in the 47-3-1 strain of the Black and White Rice Transformant expressing the IFS2 gene under Glb promoter control at a time coinciding with the retention time of Zenithine standard sample. It can be seen that the synthesis (Fig. 5C). As a result of HPLC / MS analysis, as shown in FIG. 5D, it was found that the m / z value of the peak corresponding to isoflavone Zenithsteine was shown.

In addition, as a result of confirming the system for synthesizing isoflavones for all the transformant lines and calculating the amount of synthesis thereof, as shown in Table 3, black and white rice used as a control did not contain isoflavones, The transformant of the present invention was found to contain isoflavones. The content of isoflavones differed according to the transformed individuals, but IFS2 Genetically Modified Rice IFS1 It was found that the amount of isoflavones synthesized was higher than that of rice transformed with the gene. This is IFS2 It is considered that the activity of IFS2 expressed by the gene is stronger.

gene system Isoflavones (μg / g) Black rice con 0 IFS1 7-1-1 10.3 7-2-1 10.3 13-5-1 10.3 14-4-1 6.2 16-4-1 4.1 18-4-1 12.4 18-4-6 14.4 24-4-1 14.4 32-4-1 10.3 IFS2 5-2-1 24.7 17-4-1 24.7 18-4-1 20.6 31-4-1 22.6 40-4-1 4.1 41-4-1 24.7 47-3-1 37.1 56-3-1 24.7 68-4-1 18.5 73-4-1 4.1 76-3-1 16.5 Nakdong Rice con 0.0 IFS1 8-3-1 14.4 5-4-1 6.2 13-3-1 2.1 20-3-1 6.2 20-4-1 6.2 25-3-1 4.1 IFS2 4-4-1 6.2 21-3-1 4.1 27-4-1 10.29

As described above, the recombinant vector of the present invention enables the synthesis of isoflavones by stably expressing isoflavone synthase in rice which does not synthesize isoflavones, thereby transforming the isoflavone containing a higher concentration than the conventional transformants. It has the effect of being able to manufacture a converter. Therefore, the recombinant vector of the present invention can be used for the purpose of the development of a new breed of transgenic crops that biosynthesize the useful material isoflavones.

Figure 1 shows a cleavage map of the recombinant vector of the present invention. (LB (left border), RB (Right border), Glb ( Glb promoter), OsCc1 (Cytochrome c promoter), IFS1 Or IFS2 ( IFS1 or IFS2 gene), TPinll (Pin-II terminator), P35S (CaMV 35S promoter), TNos (Nos terminator), bar ( bar coding region), MAR (Matrix attachment region)

2 is confirmed by the colony PCR for transforming the Agrobacterium tumefaciens LBA4404 transformed with the recombinant vector of the present invention. (M: size marker, lane 1 to 5: A (IFS1 / GB / LBA4404 ) , B (IFS2 / GB / LBA4404 ), C (IFS1 / GGB / LBA4404 ), D (IFS2 / GGB / LBA4404 ), lanes 6 to 10: A (IFS2cDNA / GB / LBA4404 ), B (IFS2gDNA / GB / LBA4404 ) , C (IFS2cDNA / GB / LBA4404 ), D (IFS2cDNA / GB / LBA4404 ))

Figure 3 shows the transformation process of rice (A: callus induction process using rice seeds, B: selection process of transformed callus, C: regeneration process of the selected callus, D: regeneration process through selection process) Purification process of Nakdong rice transformed with IFS1 and IFS2 under the control of E and F: Glb promoter, and black rice transformed with IFS1 and IFS2 under the control of G and H: Glb promoter)

4 shows the transformation and expression of transgenic rice into which IFS1 and IFS2 genes are introduced by PCR (A), RT-PCR (B) and Southern blot analysis (C). (TG1 to TG10: respective traits) Conversion number)

Figure 5 is analyzed by LC / MS of isoflavone Zenithine in control and transformed rice.

<110> Republic of Korea <120> Recombinant vector comprising polynucleotide encoding isoflavone          synthase and transformants transformed thereby <130> P07-0100 <160> 8 <170> KopatentIn 1.71 <210> 1 <211> 1904 <212> DNA <213> Glycine max <220> <221> 5'UTR (222) (1) .. (60) <220> <221> 3'UTR (222) (1761) .. (1904) <220> <221> intron (222) (961) .. (1096) <400> 1 aacctcactc aaactcggga tcacagaaac caacaacagt tcttgcactg aggtttcacg 60 atgttgctgg aacttgcact tggtttgttt gtgttagctt tgtttctgca cttgcgtccc 120 acaccaagtg caaaatcaaa agcacttcgc cacctcccaa accctccaag cccaaagcct 180 cgtcttccct tcattggcca ccttcacctc ttaaaagata aacttctcca ctatgcactc 240 atcgatctct ccaaaaagca tggcccctta ttctctctct ccttcggctc catgccaacc 300 gtcgttgcct ccacccctga gttgttcaag ctcttcctcc aaacccacga ggcaacttcc 360 ttcaacacaa ggttccaaac ctctgccata agacgcctca cttacgacaa ctctgtggcc 420 atggttccat tcggacctta ctggaagttc gtgaggaagc tcatcatgaa cgaccttctc 480 aacgccacca ccgtcaacaa gctcaggcct ttgaggaccc aacagatccg caagttcctt 540 agggttatgg cccaaagcgc agaggcccag aagccccttg acgtcaccga ggagcttctc 600 aaatggacca acagcaccat ctccatgatg atgctcggcg aggctgagga gatcagagac 660 atcgctcgcg aggttcttaa gatcttcggc gaatacagcc tcactgactt catctggcct 720 ttgaagtatc tcaaggttgg aaagtatgag aagaggattg atgacatctt gaacaagttc 780 gaccctgtcg ttgaaagggt catcaagaag cgccgtgaga tcgtcagaag gagaaagaac 840 ggagaagttg ttgagggcga ggccagcggc gtcttcctcg acactttgct tgaattcgct 900 gaggacgaga ccatggagat caaaattacc aaggagcaaa tcaagggcct tgttgtcgac 960 agtttcctgc ttcattcatt gatcgaaata tgcagtattt tgttaacaag agatcgagaa 1020 ttgacattta tatattcatg tggtggcaat taattaacgg tacgcattct taatcgatat 1080 tgtgtatgtg caggactttt tctctgcagg gacagattcc acagcggtgg caacagagtg 1140 ggcattggca gagctcatca acaatcccag ggtgttgcaa aaggctcgtg aggaggtcta 1200 cagtgttgtg ggcaaagata gactcgttga cgaagttgac actcaaaacc ttccttacat 1260 tagggccatt gtgaaggaga cattccgaat gcacccacca ctcccagtgg tcaaaagaaa 1320 gtgcacagaa gagtgtgaga ttaatgggta tgtgatccca gagggagcat tggttctttt 1380 caatgtttgg caagtaggaa gggaccccaa atactgggac agaccatcag aattccgtcc 1440 cgagaggttc ttagaaactg gtgctgaagg ggaagcaggg cctcttgatc ttaggggcca 1500 gcatttccaa ctcctcccat ttgggtctgg gaggagaatg tgccctggtg tcaatttggc 1560 tacttcagga atggcaacac ttcttgcatc tcttatccaa tgctttgacc tgcaagtgct 1620 gggccctcaa ggacaaatat tgaaaggtga tgatgccaaa gttagcatgg aagagagagc 1680 tggcctcaca gttccaaggg cacatagtct cgtttgtgtt ccacttgcaa ggatcggcgt 1740 tgcatctaaa ctcctttctt aattaagata atcatcatat acaatagtag tgtcttgcca 1800 tcgcagttgc tttttatgta ttcataatca tcatttcaat aaggtgtgac tggtacttaa 1860 tcaagtaatt aaggttacat acatgcaaaa aaaaaaaaaa aaaa 1904 <210> 2 <211> 521 <212> PRT <213> Glycine max <400> 2 Met Leu Leu Glu Leu Ala Leu Gly Leu Phe Val Leu Ala Leu Phe Leu   1 5 10 15 His Leu Arg Pro Thr Pro Ser Ala Lys Ser Lys Ala Leu Arg His Leu              20 25 30 Pro Asn Pro Pro Ser Pro Lys Pro Arg Leu Pro Phe Ile Gly His Leu          35 40 45 His Leu Leu Lys Asp Lys Leu Leu His Tyr Ala Leu Ile Asp Leu Ser      50 55 60 Lys Lys His Gly Pro Leu Phe Ser Leu Ser Phe Gly Ser Met Pro Thr  65 70 75 80 Val Val Ala Ser Thr Pro Glu Leu Phe Lys Leu Phe Leu Gln Thr His                  85 90 95 Glu Ala Thr Ser Phe Asn Thr Arg Phe Gln Thr Ser Ala Ile Arg Arg             100 105 110 Leu Thr Tyr Asp Asn Ser Val Ala Met Val Pro Phe Gly Pro Tyr Trp         115 120 125 Lys Phe Val Arg Lys Leu Ile Met Asn Asp Leu Leu Asn Ala Thr Thr     130 135 140 Val Asn Lys Leu Arg Pro Leu Arg Thr Gln Gln Ile Arg Lys Phe Leu 145 150 155 160 Arg Val Met Ala Gln Ser Ala Glu Ala Gln Lys Pro Leu Asp Val Thr                 165 170 175 Glu Glu Leu Leu Lys Trp Thr Asn Ser Thr Ile Ser Met Met Met Leu             180 185 190 Gly Glu Ala Glu Glu Ile Arg Asp Ile Ala Arg Glu Val Leu Lys Ile         195 200 205 Phe Gly Glu Tyr Ser Leu Thr Asp Phe Ile Trp Pro Leu Lys Tyr Leu     210 215 220 Lys Val Gly Lys Tyr Glu Lys Arg Ile Asp Asp Ile Leu Asn Lys Phe 225 230 235 240 Asp Pro Val Val Glu Arg Val Ile Lys Lys Arg Arg Glu Ile Val Arg                 245 250 255 Arg Arg Lys Asn Gly Glu Val Val Glu Gly Glu Ala Ser Gly Val Phe             260 265 270 Leu Asp Thr Leu Leu Glu Phe Ala Glu Asp Glu Thr Met Glu Ile Lys         275 280 285 Ile Thr Lys Glu Gln Ile Lys Gly Leu Val Val Asp Phe Phe Ser Ala     290 295 300 Gly Thr Asp Ser Thr Ala Val Ala Thr Glu Trp Ala Leu Ala Glu Leu 305 310 315 320 Ile Asn Asn Pro Arg Val Leu Gln Lys Ala Arg Glu Glu Val Tyr Ser                 325 330 335 Val Val Gly Lys Asp Arg Leu Val Asp Glu Val Asp Thr Gln Asn Leu             340 345 350 Pro Tyr Ile Arg Ala Ile Val Lys Glu Thr Phe Arg Met His Pro Pro         355 360 365 Leu Pro Val Val Lys Arg Lys Cys Thr Glu Glu Cys Glu Ile Asn Gly     370 375 380 Tyr Val Ile Pro Glu Gly Ala Leu Val Leu Phe Asn Val Trp Gln Val 385 390 395 400 Gly Arg Asp Pro Lys Tyr Trp Asp Arg Pro Ser Glu Phe Arg Pro Glu                 405 410 415 Arg Phe Leu Glu Thr Gly Ala Glu Gly Glu Ala Gly Pro Leu Asp Leu             420 425 430 Arg Gly Gln His Phe Gln Leu Leu Pro Phe Gly Ser Gly Arg Arg Met         435 440 445 Cys Pro Gly Val Asn Leu Ala Thr Ser Gly Met Ala Thr Leu Leu Ala     450 455 460 Ser Leu Ile Gln Cys Phe Asp Leu Gln Val Leu Gly Pro Gln Gly Gln 465 470 475 480 Ile Leu Lys Gly Asp Asp Ala Lys Val Ser Met Glu Glu Arg Ala Gly                 485 490 495 Leu Thr Val Pro Arg Ala His Ser Leu Val Cys Val Pro Leu Ala Arg             500 505 510 Ile Gly Val Ala Ser Lys Leu Leu Ser         515 520 <210> 3 <211> 1949 <212> DNA <213> Glycine max <220> <221> 5'UTR (222) (1) .. (48) <220> <221> 3'UTR (222) (1766) .. (1949) <220> <221> intron (222) (961) .. (1096) <400> 3 attagcctca caaaagcaaa gatcaaacaa accaaggacg agaacacgat gttgcttgaa 60 cttgcacttg gtttattggt tttggctctg tttctgcact tgcgtcccac acccactgca 120 aaatcaaaag cacttcgcca actcccaaac ccaccaagcc caaagcctcg tcttcccttc 180 ataggacacc ttcatctctt aaaagacaaa cttctccact acgcactcat cgacctctcc 240 aaaaaacatg gtcccttatt ctctctctac tttggctcca tgccaaccgt tgttgcctcc 300 acaccagaat tgttcaagct cttcctccaa acgcacgagg caacttcctt caactcaagg 360 ttccaaacct cagccataag acgcctcacc tatgatagct cagtggccat ggttcccttc 420 ggaccttact ggaagttcgt gaggaagctc atcatgaacg accttctcaa cgccaccact 480 gtaaacaagt tgaggccttt gaggacccaa cagatccgca agttccttag ggttatggcc 540 caaggcgcag aggcacagaa gccccttgac ttgaccgagg agcttctgaa atggaccaac 600 agcaccatct ccatgatgat gctcggcgag gctgaggaga tcagagacat cgctcgcgag 660 gttcttaaga tctttggcga atacagcctc actgacttca tctggccatt gaagcatctc 720 aaggttggaa agtatgagtc aagaagcgcc gtgaagagga tcgacgacat cttgaacaag 780 ttcgaccctg tcgttgaaag ggtcatcaag aagcgccgtg agatcgtgag gaggagaaag 840 aacggagagg ttgttgaggg tgaggtcagc ggggttttcc ttgacacttt gcttgaattc 900 gctgaggatg agaccatgga gatcaaaatc accaaggacc acatcaaggg tcttgttgtc 960 gtgagtttcc tgcttcattc attgatcgaa atatgcagta ttttgttaac aagagatcga 1020 gaattgacat ttatatattc atgtggtggc aattaattaa cggtacgcat tcttaatcga 1080 tattgtgtat gtgcaggact ttttctcggc aggaacagac tccacagcgg tggcaacaga 1140 gtgggcattg gcagaactca tcaacaatcc taaggtgttg gaaaaggctc gtgaggaggt 1200 ctacagtgtt gtgggaaagg acagacttgt ggacgaagtt gacactcaaa accttcctta 1260 cattagagca atcgtgaagg agacattccg catgcacccg ccactcccag tggtcaaaag 1320 aaagtgcaca gaagagtgtg agattaatgg atatgtgatc ccagagggag cattgattct 1380 cttcaatgta tggcaagtag gaagagaccc caaatactgg gacagaccat cggagttccg 1440 tcctgagagg ttcctagaga caggggctga aggggaagca gggcctcttg atcttagggg 1500 acaacatttt caacttctcc catttgggtc tgggaggaga atgtgccctg gagtcaatct 1560 ggctacttcg ggaatggcaa cacttcttgc atctcttatt cagtgcttcg acttgcaagt 1620 gctgggtcca caaggacaga tattgaaggg tggtgacgcc aaagttagca tggaagagag 1680 agccggcctc actgttccaa ggtcacatag tcttgtctgt gttccacttg caaggatcgg 1740 cgttgcatct aaactccttt cttaattaag atcatcatca tatataatat ttactttttg 1800 tgtgttgata atcatcattt caataaggtc tcgttcatca actttttatg aagtatataa 1860 gcccttccat gcacattgta tcatctccca tttgtcttcg tttgcaatca ctagtgaatt 1920 cgcggccgcc tgcaggtcga ccatatggg 1949 <210> 4 <211> 526 <212> PRT <213> Glycine max <400> 4 Met Leu Leu Glu Leu Ala Leu Gly Leu Leu Val Leu Ala Leu Phe Leu   1 5 10 15 His Leu Arg Pro Thr Pro Thr Ala Lys Ser Lys Ala Leu Arg Gln Leu              20 25 30 Pro Asn Pro Pro Ser Pro Lys Pro Arg Leu Pro Phe Ile Gly His Leu          35 40 45 His Leu Leu Lys Asp Lys Leu Leu His Tyr Ala Leu Ile Asp Leu Ser      50 55 60 Lys Lys His Gly Pro Leu Phe Ser Leu Tyr Phe Gly Ser Met Pro Thr  65 70 75 80 Val Val Ala Ser Thr Pro Glu Leu Phe Lys Leu Phe Leu Gln Thr His                  85 90 95 Glu Ala Thr Ser Phe Asn Ser Arg Phe Gln Thr Ser Ala Ile Arg Arg             100 105 110 Leu Thr Tyr Asp Ser Ser Val Ala Met Val Pro Phe Gly Pro Tyr Trp         115 120 125 Lys Phe Val Arg Lys Leu Ile Met Asn Asp Leu Leu Asn Ala Thr Thr     130 135 140 Val Asn Lys Leu Arg Pro Leu Arg Thr Gln Gln Ile Arg Lys Phe Leu 145 150 155 160 Arg Val Met Ala Gln Gly Ala Glu Ala Gln Lys Pro Leu Asp Leu Thr                 165 170 175 Glu Glu Leu Leu Lys Trp Thr Asn Ser Thr Ile Ser Met Met Met Leu             180 185 190 Gly Glu Ala Glu Glu Ile Arg Asp Ile Ala Arg Glu Val Leu Lys Ile         195 200 205 Phe Gly Glu Tyr Ser Leu Thr Asp Phe Ile Trp Pro Leu Lys His Leu     210 215 220 Lys Val Gly Lys Tyr Glu Ser Arg Ser Ala Val Lys Arg Ile Asp Asp 225 230 235 240 Ile Leu Asn Lys Phe Asp Pro Val Val Glu Arg Val Ile Lys Lys Arg                 245 250 255 Arg Glu Ile Val Arg Arg Arg Lys Asn Gly Glu Val Val Glu Gly Glu             260 265 270 Val Ser Gly Val Phe Leu Asp Thr Leu Leu Glu Phe Ala Glu Asp Glu         275 280 285 Thr Met Glu Ile Lys Ile Thr Lys Asp His Ile Lys Gly Leu Val Val     290 295 300 Asp Phe Phe Ser Ala Gly Thr Asp Ser Thr Ala Val Ala Thr Glu Trp 305 310 315 320 Ala Leu Ala Glu Leu Ile Asn Asn Pro Lys Val Leu Glu Lys Ala Arg                 325 330 335 Glu Glu Val Tyr Ser Val Val Gly Lys Asp Arg Leu Val Asp Glu Val             340 345 350 Asp Thr Gln Asn Leu Pro Tyr Ile Arg Ala Ile Val Lys Glu Thr Phe         355 360 365 Arg Met His Pro Pro Leu Pro Val Val Lys Arg Lys Cys Thr Glu Glu     370 375 380 Cys Glu Ile Asn Gly Tyr Val Ile Pro Glu Gly Ala Leu Ile Leu Phe 385 390 395 400 Asn Val Trp Gln Val Gly Arg Asp Pro Lys Tyr Trp Asp Arg Pro Ser                 405 410 415 Glu Phe Arg Pro Glu Arg Phe Leu Glu Thr Gly Ala Glu Gly Glu Ala             420 425 430 Gly Pro Leu Asp Leu Arg Gly Gln His Phe Gln Leu Leu Pro Phe Gly         435 440 445 Ser Gly Arg Arg Met Cys Pro Gly Val Asn Leu Ala Thr Ser Gly Met     450 455 460 Ala Thr Leu Leu Ala Ser Leu Ile Gln Cys Phe Asp Leu Gln Val Leu 465 470 475 480 Gly Pro Gln Gly Gln Ile Leu Lys Gly Gly Asp Ala Lys Val Ser Met                 485 490 495 Glu Glu Arg Ala Gly Leu Thr Val Pro Arg Ser His Ser Leu Val Cys             500 505 510 Val Pro Leu Ala Arg Ile Gly Val Ala Ser Lys Leu Leu Ser         515 520 525 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> IFS1FOR <400> 5 gtaattaacc tcactcaaac tcgg 24 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> IFS1REV <400> 6 gcaaacgaag acaaatggga gatgata 27 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> IFS2FOR <400> 7 aaaattagcc tcacaaaagc aaag 24 <210> 8 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> IFS2REV <400> 8 gcaaacgaag acaaatggga gatgata 27  

Claims (8)

(a) preparing a pMJC-GB-IFS2 or pMJC-GGB-IFS2 recombinant vector comprising a polynucleotide encoding IFS2 of SEQ ID NO: 3; And (b) transforming rice with the recombinant vector; Method for producing a transformed rice producing an isoflavone comprising a. (a) preparing a pMJC-GB-IFS2 or pMJC-GGB-IFS2 recombinant vector comprising a polynucleotide encoding IFS2 of SEQ ID NO: 3; (b) transforming rice with the recombinant vector; And (c) extracting isoflavones from the transformed rice Method for producing isoflavones from rice comprising a. delete delete delete delete delete delete

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