KR101399944B1 - Method for producing transgenic soybean plant with improved syringin and coniferin content and transgenic soybean plant produced by the same - Google Patents

Abstract

The present invention relates to a method of increasing the content of syringin and coniferin in a soybean plant compared to a wild form by transforming soybean plant cells with a recombinant vector including a gene coding the recombinant glycosyltransferase UGT72E-3/2 protein, a method for producing transgenic soybean plants with an increased content of syringin and coniferin compared to the wild form, transgenic soybean plants with an increased content of syringin and coniferin compared to the wild form produced thereby and seeds thereof, and a composition for increasing the content of syringin and coniferin in soybean plants including the recombinant vector as an active ingredient. When a gene coding the recombinant glycosyltransferase UGT72E-3/2 protein of the present invention is used, soybean plants containing a large quantity of syringin and coniferin which are not produced in general wild form soybeans can be produced. Accordingly, soybeans with high functionality and high quality are able to be produced, thereby expected to be useful in revitalizing the soybean food industry.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method for producing transgenic soybean plants having increased syringine and conipherin contents,

The present invention relates to a method for producing a transgenic soybean plant having increased syringine and conipherin contents and a soybean plant according to the method, and more particularly, to a method for producing a recombinant vector containing a gene encoding a recombinant glycoprotein UGT72E-3/2 protein A method of increasing the content of syringine and coniferin in soybean plants by transforming soybean plant cells with wild type, a method of producing transgenic soybean plants having increased syringin and conipherin content compared to wild type, a method of producing wild type And a composition for increasing the content of syringine and coniferin in soybean plants containing the recombinant vector as an active ingredient.

Soy is a well-known dapper crop, which can be grown under various conditions and is cultivated all over the world. Beans are rich in grains, 40% protein rich in nutrients, 60% protein and fat, and are the most unique crops with the highest protein and fat content except processed foods such as cheese. Especially, Asian people with rice stocks are involved in economical protein causes and secondary metabolites, so they are constantly exploring varietal development techniques including transformation to improve functional aspects of soybean.

Primary metabolites are carbohydrates, proteins, nucleic acids and lipids, which are indispensable for performing cellular functions. On the other hand, there are many compounds in plants that are not essential for survival, which are called secondary metabolites. Secondary metabolites can be divided into alkaloids, terpenes, phenolic compounds, and other compounds depending on the structure and synthesis process.

The syringin is called Siberian ginseng ( Eleutherococcus senticosus Maxim ) is a pharmacological component that has excellent mental and physical adaptability to stress. It is classified as a typical adaptogen originating from plants. An adaptogen is a plant secondary metabolite that responds to various stresses and increases the non-specific resistance of a living organism without side effects. It is also known as Eleutheroside B, which is a lignin glycoside produced by the synthesis of phenylpropanoid, which is a component of lignin, sinapyl alcohol (s type monolignol) (UDP-glucose transferase). It has been known that sylindin has a strong physiological activity in antistress, antimicrobial and the like together with Ereotideoside E. In particular, recently, the purely isolated seuljin has been reported to exhibit excellent efficacy in the treatment of diabetes and depression, which are the most important health problems of modern urban people.

In the meantime, the soybean transformation in Korea has been insufficient in the regeneration technology of soybean plant through tissue culture, and unlike other crops, it is difficult to introduce foreign gene, so a stable transformation technology system has not yet been established. In some laboratories, there were partial successes, but failed to produce sustained results. As a result of visiting and studying the US and Japan, the present inventors produced herbicide-resistant transgenic soybean as domestic varieties in 2006 with its own developed technology, and now it is possible to mass-produce soybean transformants using domestic digestive useful genes.

Soybeans are most commonly consumed in the form of bean processed foods, often known as soy bean sprouts (33.2%), soy sauce (31.2%), tofu (26.6%) and soy milk (9.0%). For the globalization of soybean-based plants and the activation of the soybean-related industry, it is considered urgent that the functional enhancement of soybeans as a raw material and the development of high-quality processing technology are urgently needed. Soybean food, known as Oriental traditional food, is a food containing a large amount of health functional ingredients, and its excellence is widely known.

Korean Patent Laid-Open Publication No. 1994-0018095 discloses an antiallergic medicinal composition containing aspirin as an active ingredient, Korean Patent Publication No. 2004-0004764 discloses a medicinal composition containing a syringine having a protecting activity against hepatotoxicity However, as described in the present invention, the method for producing transgenic soybean plants having increased amounts of syringin and conipherin and the resulting soybean plants have not been disclosed.

Disclosure of the Invention The present invention has been made in view of the above needs, and the present inventors have found that a gene encoding a recombinant herpes simplex protein UGT72E-3/2 is regulated by a 35S promoter or a β-conglycinin promoter The present inventors have completed the present invention by confirming the increase in the amount of syringine and conipherin in the leaves of the soybean plants transformed with the recombinant vector.

In order to solve the above problems, the present invention provides a recombinant vector comprising a gene encoding a recombinant glycosyltransferase UGT72E-3/2 protein comprising the amino acid sequence of SEQ ID NO: 2 to transform soybean plant cells into UGT72E-3/2 The present invention provides a method for increasing syringin and coniferin content in soybean plants compared to the wild type comprising overexpressing the gene.

In addition, the present invention relates to a method for producing a recombinant vector, which comprises transforming a soybean plant cell with a recombinant vector comprising a gene encoding the recombinant herpes simplex protein UGT72E-3/2, A method for producing a bean plant is provided.

In addition, the present invention provides a transformed soybean plant having increased syringine and coniferin content compared to the wild type strain produced by the above method, and seeds of the soybean plant.

The present invention also provides a composition for increasing the content of syringine and coniferin in soybean plants containing a recombinant vector comprising a gene encoding a recombinant glycosyltransferase UGT72E-3/2 protein comprising the amino acid sequence of SEQ ID NO: 2 as an active ingredient to provide.

According to the present invention, it is possible to produce a soybean plant containing a large amount of choline chloride and conipherin, which is not produced in general wild type soybean, by using the recombinant herbicide UGT72E-3/2 protein coding gene, It is expected to be useful for the activation of the soybean food industry.

Figure 1 shows two recombinant vectors (35S-UGT72E-3/2 and &bgr; -UGT72E-3/2) containing the gene encoding the recombinant glycosyltransferase UGT72E-3/2 protein using the 35S promoter and the β- conglycine promoter ). ≪ / RTI >
Fig. 2 shows the growth process of soybean plants transformed with recombinant vectors 35S-UGT72E-3/2 and? -UGT72E-3/2.
Figure 3 shows the results of PCR performed to confirm the inserted gene from the 35S-UGT72E-3/2 T 0 transformant and β-UGT72E-3/2 T 0 transformants. M: Marker, CON: wild type light bean, 1-7: 35S-UGT72E-3/2 transformant, 8-14: β-UGT72E-3/2 transformant.
Figure 4 illustrates a 35S-UGT72E-3/2 T 0 transformant and β-UGT72E-3/2 T 0 transformants was performed to confirm the inserted gene from the transformants by RT-PCR results. M: Marker, CON: wild type light bean, 1-7: 35S-UGT72E-3/2 transformant, 8-14: β-UGT72E-3/2 transformant. (9 is the result of PCR in Fig. 3, and the gene introduction is not confirmed and is excluded from the RT-PCR target)
Leaves of Figure 5 is the wild-type light stabilizer beans and 35S-UGT72E-3/2 T 0 transformants (35s-ugt # 7), and β-UGT72E-3/2 T 0 transformants (β-ugt # 3, 7 ) And the results of HPLC analysis of the content of syringine and conipherin in the sample.

In order to accomplish the object of the present invention, the present invention provides a recombinant vector comprising a gene encoding the recombinant glycosyltransferase UGT72E-3/2 protein comprising the amino acid sequence of SEQ ID NO: 2, / 2 < / RTI > gene of the wild type, which comprises the step of overexpressing the syringin and coniferin in the soybean plant.

In the method according to one embodiment of the present invention, the recombinant herpes simplex enzyme UGT72E-3/2 The protein may comprise, but is not limited to, the amino acid sequence of SEQ ID NO: 2.

The recombinant herpetous enzyme UGT72E-3/2 according to the present invention The range of the protein includes a protein having the amino acid sequence represented by SEQ ID NO: 2 and a functional equivalent of the protein. Is at least 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 90% or more, more preferably 90% or more, Quot; refers to a protein having a homology of at least 95% with a physiological activity substantially equivalent to that of the protein represented by SEQ ID NO: 2. "Substantially homogenous physiological activity" means an activity that increases the syringin and conipherin content in a plant.

In the method according to one embodiment of the present invention, the recombinant herpes simplex enzyme UGT72E-3/2 The gene coding for the protein may consist of the nucleotide sequence of SEQ ID NO: 1, but is not limited thereto. In addition, homologues of the nucleotide sequences are included within the scope of the present invention. Specifically, the gene has a nucleotide sequence having a sequence homology of 70% or more, more preferably 80% or more, still more preferably 90% or more, and most preferably 95% or more, with the nucleotide sequence of SEQ ID NO: 1 . "% Of sequence homology to polynucleotides" is ascertained by comparing the comparison region with two optimally aligned sequences, and a portion of the polynucleotide sequence in the comparison region is the reference sequence for the optimal alignment of the two sequences (I. E., A gap) relative to the < / RTI >

The recombinant vector of the present invention may include a promoter and a gene encoding a UGT72E-3/2 protein, and may further include a terminator, a selection marker, and the like.

The promoter may be CaMV 35S, β-conglycinin, actin, ubiquitin, pEMU, MAS or a histone promoter, preferably a 35S-promoter and a β-conglycinin promoter , But is not limited thereto.

The term "recombinant" refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, heterologous peptide or heterologous nucleic acid. The recombinant cell can express a gene or a gene fragment that is not found in the natural form of the cell in one of the sense or antisense form. In addition, the recombinant cell can express a gene found in a cell in its natural state, but the gene has been modified and reintroduced intracellularly by an artificial means.

In the present invention, the UGT72E-3/2 gene sequence can be inserted into a recombinant expression vector. The term "recombinant expression vector" means a bacterial plasmid, a phage, a yeast plasmid, a plant cell virus, a mammalian cell virus, or other vector. In principle, any plasmid and vector can be used if it can replicate and stabilize within the host. An important characteristic of the expression vector is that it has a replication origin, a promoter, a marker gene and a translation control element.

Expression vectors containing the UGT72E-3/2 gene sequence and appropriate transcription / translation control signals can be constructed by methods known to those skilled in the art. Such methods include in vitro recombinant DNA technology, DNA synthesis techniques, and in vivo recombination techniques. The DNA sequence can be effectively linked to appropriate promoters in the expression vector to drive mRNA synthesis. The expression vector may also include a ribosome binding site and a transcription terminator as a translation initiation site.

A preferred example of the recombinant vector of the present invention is a Ti-plasmid vector capable of transferring a so-called T-region to a plant cell when present in a suitable host such as Agrobacterium tumefaciens . Other types of Ti-plasmid vectors (see EP 0 116 718 B1) are currently used to transfer hybrid DNA sequences to plant cells or protoplasts in which new plants capable of properly inserting hybrid DNA into the plant's genome can be produced have. A particularly preferred form of the Ti-plasmid vector is a so-called binary vector as claimed in EP 0 120 516 B1 and U.S. Patent No. 4,940,838. Other suitable vectors that can be used to introduce the DNA according to the invention into the plant host include viral vectors such as those that can be derived from double-stranded plant viruses (e. G., CaMV) and single- For example, from non -complete plant virus vectors. The use of such vectors may be particularly advantageous when it is difficult to transform the plant host properly.

The expression vector preferably comprises one or more selectable markers. The marker is typically a nucleic acid sequence having a property that can be selected by a chemical method, and includes all genes capable of distinguishing a transformed cell from a non-transformed cell. Examples include herbicide resistance genes such as glyphosate or phosphinothricin, antibiotics such as kanamycin, G418, Bleomycin, hygromycin, chloramphenicol, Resistant genes, but are not limited thereto.

In the recombinant vector of the present invention, conventional terminators can be used. Examples thereof include nopaline synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, Agrobacterium and the terminator of the Octopine gene of Tumefaciens, but the present invention is not limited thereto. Regarding the need for terminators, it is generally known that such regions increase the certainty and efficiency of transcription in plant cells. The use of terminators is therefore highly desirable in the context of the present invention.

Any host cell known in the art may be used as the host cell capable of continuously cloning and expressing the vector of the present invention in a stable and prokaryotic cell, for example, E. coli JM109, E. coli BL21, E. coli RR1 , E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus tulifene, and Salmonella typhimurium, Serratia marcesensus and various Pseudomonas species And enterobacteria and strains such as the species.

When the vector of the present invention is transformed into eukaryotic cells, yeast ( Saccharomyce cerevisiae), and the like insect cells, human cells (e.g., CHO cells (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cell lines) and plant cell may be used. The host cell is preferably a plant cell.

The method of delivering the vector of the present invention into a host cell can be carried out by the CaCl2 method, Hanahan, D., J. MoI. Biol., 166: 557-580 (1983) An electric drilling method or the like. When the host cell is a eukaryotic cell, the vector is injected into the host cell by microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, gene bombardment or the like .

In addition, the present invention relates to a promoter and Arabidopsis The present invention relates to a method for producing a recombinant vector, which comprises transforming a plant cell with a recombinant vector comprising a UGT72E2 gene and a UGT72E3 gene obtained by recombining a UGT72E2 gene and a UGT72E3 gene derived from a transgenic plant cell and regenerating the plant from the transformed plant cell, The present invention provides a method of producing a plant having increased gin and coniferin content.

The method of the invention comprises the step of transforming a plant cell with a recombinant vector according to the present invention, the transformant is, for example, Agrobacterium tumefaciens may be mediated by (Agrobacterium tumefiaciens). In addition, the method of the present invention comprises regenerating a transgenic plant from the transformed plant cell. Any of the methods known in the art can be used for regeneration of transgenic plants from transgenic plant cells.

Transformed plant cells must be regenerated into whole plants. Techniques for the regeneration of mature plants from callus or protoplast cultures are well known in the art for a number of different species (Handbook of Plant Cell Culture, Vol. 1-5, 1983-1989, Momillan, N. Y.).

In addition, the present invention provides transgenic plants and seeds thereof having increased syringin and coniferin content compared to the wild type produced by the above method. Preferably, the plant is selected from the group consisting of Arabidopsis, potato, eggplant, cigarette, pepper, tomato, burdock, ciliaceae, lettuce, bellflower, spinach, modern sweet potato, celery, carrot, parsley, parsley, cabbage, cabbage, Rice, barley, wheat, rye, corn, sorghum, oats, onion and the like, preferably dicotyledonous plants such as rice, barley, wheat, rye, corn, Plant, and more preferably, the dicotyledonous plant is soybean ( Glycine max ), but is not limited thereto.

The present invention also provides a recombinant vector comprising a gene encoding a recombinant glycosyltransferase UGT72E-3/2 protein comprising the amino acid sequence of SEQ ID NO: 2, wherein the recombinant vector comprises syringin and coniferin, Thereby providing a transgenic soybean plant having an increased content.

The present invention also relates to a composition for increasing the content of sirolin and coniferin of a soybean plant containing a recombinant vector comprising a gene encoding a recombinant glycosyltransferase UGT72E-3/2 protein comprising the amino acid sequence of SEQ ID NO: 2 as an active ingredient to provide. The composition of the present invention comprises, as an active ingredient, a recombinant vector comprising a gene encoding a recombinant glycosyltransferase UGT72E-3/2 protein comprising the amino acid sequence of SEQ ID NO: 2 and transforming the recombinant vector into a plant, Can increase the content of syringine and conipherin in the body.

Hereinafter, 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.

Experimental Method

Production of soybean transformation vector

Recombinant glycoprotein UGT72E-3/2 protein coding gene was purchased from Prof. Nam Jae-sung of Dong-A University and used for the present invention.

First of all, it has been reported that the enzyme UGT72E, which has been reported to have the ability to transfer sugar to cinnamyl alcohol or structurally similar coniferyl alcohol, Enzymatic properties were investigated. The UGT72E family also has structural similarity to that of a general glycoprotein, with the amino terminal region having a substrate recognition region and the carboxy terminal having an enzymatic active region that transfers the sugar from the sugar activated by UDP to the substrate, Carboxy-terminal plant secondary product glucosyltransferase (PSPG) motifs have been reported to be important for the activity of plant-derived glycosyltransferases. In the UGT72E family, there are UGT72E1, UGT72E2 and UGT72E3, which have similar nucleotide sequences.

In the present invention, UGT72E2 and UGT72E3 were each subdivided into an amino fragment containing amino acids 1 to 344 at the amino terminus and a carboxy fragment containing the amino acid at position 345 to 481 of the carboxy terminus. The amino fragment contains a region that determines the substrate recognition specificity and the carboxy terminal contains PSPG motifs important for glycosylation. In order to efficiently produce the syringine in the plant, the substrate specificity is more important than the glycoprotein activity. Therefore, the amino fragment is enlarged to about 3/4 of the whole, and the carboxy fragment is divided into the minimum size including the PSPG motif. As a result, the recombinant UGT72E-3/2 gene of the present invention contains an amino fragment containing amino acids 1 to 344 at the amino terminus of UGT72E3 and an amino acid fragment containing amino acids from 345 of UGT72E2 to 481 of the carboxy terminus Lt; RTI ID = 0.0 > fragments. ≪ / RTI >

The recombinant recombinant herpesvirus UGT72E-3/2 protein coding gene was ligated with pB2GW7.0 and pB2GW7.0- (the 35S promoter was replaced with a β-conglycinin promoter, which is a destination vector) ) Using Invitrogen's Gateway LR Clonase (TM) Enzyme mix Kit. Subsequently, DH5? Of Invitrogen was transformed with competent cells. The pB2GW7.0-UGT72E-3/2 and pB2GW7.0-p-UGT72E-3/2 vectors prepared above were obtained from Agrobacterium tumefaciens ) EHA105.

Soybean transformant production and T1  Seed production

Seed disinfection and Immersion

The seeds were sterilized with chlorine gas generated by mixing KCl (12 N HCl 5 ml) and lactose (12% sodium hypochlorite 100 ml) for 20 hours and then mixed with 1% sodium hypochlorite for 10 minutes Respectively. And washed three times with sterilized water every 10 minutes. The sterilized seeds were placed in a 50 ml tube, and sterilized water was poured therein, followed by soaking at room temperature for 20 hours.

Agrobacterium tumefaciens ( Agrobacterium tumefaciens ) Preparation

Agrobacterium tumefaciens strain EHA105 into which pB2GW7.0-UGT72E-3/2 and pB2GW7.0-p-UGT72E-3/2 vectors were inserted was used. The strain was cultured in solid YEP medium (spectinomycin 50 mg / L, rifampicin 25 mg / L, peptone 10 g / L, sodium chloride 5 g / L, yeast extract 5 g / L, agar 1.5% After streaking, the resulting single colony was incubated at 25 ° C, and the resulting solution was added to 10 ml of a liquid YEP medium containing the same antibiotic and stirred at 25 ° C at 250 rpm until it reached OD ₆₅₀ 0.8. 10 ml of a 30% glycerol stock solution was vortexed, and 1 ml of the solution was added to a 1.5 ml tube, followed by rapid cooling with liquid nitrogen, followed by storage at -70 ° C. One day before the inoculation, 1 ml of the stored Agrobacterium tumefaciens (competent cells) was added to 200 ml of a liquid YEP medium containing antibiotics and stirred at 250 rpm at 25 ° C until the OD ₆₅₀ reached 0.8 to 1.0 .

On the day of the inoculation, 200 ml of liquid YEP medium was divided into 50 ml each, and centrifugation was carried out at 20 ° C and 3,270 x g for 10 minutes. The Agrobacterium tumefaciens pellet in each tube was suspended in a liquid co-culture medium (B5 salt 0.32 g / l, benzyladenine 1.67 mg / l, MES (N-morpholinoethane sulphonic acid) 20 mM, gibberellic acid 0.25 mg / L, acetosyringone 0.2 mM, L-cysteine 3.3 mM, sodium thiosulfate 1.0 mM, DTT 1.0 mM, sucrose 3%, pH 5.4). The high concentration concentrate was prepared by adding 5 mL of a liquid co-culture medium and 15 mL of a liquid co-culture medium at a low concentration.

Inoculation and Co-culture

The scalpel was inserted between the cotyledons of the soaked seeds, and the seeds were cut vertically until the hypocotyl was removed. Hypocotyl was cut at a distance of about 1 cm under the cotyledon, and the affected side was wound with a surgical knife (# 11 blade) about 8-10 times. At this time, 5 ml concentrate was applied to the surgical knife, and the target area was wounded. Approximately 50 explants were placed in a tube with 15 ml concentrate, sonicated for 20 seconds, vacuumed for 30 seconds with a desiccator and diaphragm pump, and inoculated for 30 minutes.

The food pieces were taken out from the tubes and placed on sterilized filter paper. After removing the water, a piece of filter paper was placed on the solid co-culture medium (same as liquid co-culture medium, agar 0.5%) and adaxial side down ). After sealing with a micropore, the cells were incubated for 5 days at 25 ° C for 18 hours.

Washing and Shinshu  Judo

After five days of co-culture, the cells were treated with liquid 1/2 hypodermic induction medium (B5 salt 1.6 g / l, benzyladenine 835 / / ℓ, MES 1.5 mM, sucrose 1.5%, cell susceptible 250 mg / ℓ, vancomycin 50 mg / l, ticarcillin 100 mg / l, pH 5.6) for 10 minutes. After the meal was placed on the filter paper and the water was removed, the shoot induction medium without selection antibiotic-① (B5 salt 3.2 g / l, benzyladenine 1.67 mg / l, MES 3 mM, agar 0.6%, sucrose 3% (5 mg / l, vancomycin 50 mg / l, ticarcillin 100 mg / l, pH 5.6) were fixed to the medium and the part to be regenerated was placed at an angle of about 30 ° C flat side up). Each plate was sealed with micropores and incubated at 25 ° C for 18 hours.

After 2 weeks, the outgrowth of the shoots was selected. The shoot induction medium containing the antibiotics-② (B5 salt 3.16g / l, MES 3mM, benzyladenine 1.67mg / l, sucrose 3%, agar 0.6% / l, DL-phosphinothricin 10 mg / l, pH 5.6). At this time, the remaining portion except for shoots was cut off and adaxial side down.

Shinshu  kidney

After 2 weeks, browned shoots and shoot pads were shaved with a surgical knife (# 15 blade) and cultured in fresh kidney medium (MS salt 4.4 g / l, MES 3 mM, gibberellic acid 0.5 mg / 1 mg / l of aspartic acid, 100 mg / l of pyroglutamic acid, 0.1 mg / l of indole acetic acid, 1 mg / l of zeatin-riboside, 3% of sucrose, 0.8% of agar, 250 mg / L, vancomycin 50 mg / L, ticacillin 100 mg / L, DL-phosphinotricin 5 mg / L, pH 5.6). Every 2 weeks, it was transferred to a new kidney kidney medium. The browning area of the shoot was removed on the top of the surgical knife (# 15 blade), and the shoot pad was gradually shaved to absorb the medium well.

Root Formation and Leaf Painting leaf painting )

When the elongated shoots were over 4 cm in length, the shoots were cut with a surgical knife (# 11 blade), and rooting medium (MS salt 4.4 g / l, MES 3 mM, asparagine 25 mg / / L, sucrose 3%, agar 0.8%, cell tubercum 50 mg / L, vancomycin 50 mg / L, ticarcillin 50 mg / L, pH 5.6). At this time, the lower part of the elongated shoot which had been cut and separated was immersed in 1 mg / ml indolebutyl acid for 3 minutes, taken out, and put in a test tube containing rooting medium. After one to two weeks have elapsed, if more than two roots are found, the medium is washed with distilled water, and a small flowerpot (6 cm x 6 cm x 5.6 cm) mixed with a 2: 1 mixture of soil (BioPrug No.2, Cm). The pots were again placed in a magenta box and grown at 25 ° C for 18 hours. After about 10 days, leaf painting was performed with 100 mg / L DL-phosphinotricin.

Seed harvest

Plants planted with large leaves (20 cm × 20 cm × 20 cm) planted with leaf painting confirmed 10 small holes in a clear plastic cover (12 cm × 12 cm × 18 cm) and plants were grown appropriately When the cover was removed, the seeds were harvested.

Confirmation of introduction of the inserted gene

PCR

Total DNA was isolated from the leaves of the transformants by the CTAB (certyltrimethyl-ammonium bromide) method. PCR was carried out using the sequence of the transgene UGT72E-3/2. The primer sequences used in the PCR are as follows: UGT72E-F 5'-GAT GAT CGC ATG GCC ACT TTT-3 '(SEQ ID NO: 3) and UGT72E-R 5'-CTA AGC ACC ACG TGA CAA GTC CAC- (SEQ ID NO: 4).

RT - PCR

Total RNA was extracted from leaves of wild type light beans and transformants using Plant RNA Purification Reagent (Invitrogen, Carlsbad, Calif.). RT-PCR was performed using Maxime RT-PCR PreMix (iNtRON, KOREA). Semi-quantitative RT-PCR was performed to examine the transcription level of the transgene, UGT72E-3/2. The primer sequences used in the RT-PCR are as follows: TuB-F 5'-TGA GCA GTT CAC GGC CAT GCT-3 '(SEQ ID NO: 5), TuB-R 5'- TCA TCC TCG GCA GTG GCA TCC T- 3 '(SEQ ID NO: 3) and UGT72E-R 5'-CTA AGC ACC ACG TGA CAA GTC CAC-3' (SEQ ID NO: 6), UGT72E-F 5'- GAT GAT CGC ATG GCC ACT TTT- 4).

Sirin Jin Coniferin  Extraction and HPLC  analysis

Root and aerial tissues were harvested from the 24-day-old plants in a liquid medium in which the medium was removed with a filter paper, in order to extract the cinnafil and coniferyl alcohol 4-O-glucoside accumulated in the plants. At 60 ° C, the tissue was dried to become a constant volume, and ground and crushed to a fine powder. 80 mg of dry aerial tissue powder (60 mg in the case of root cells) is left to stand at room temperature for 14 hours with 1.5 ml of methanol. After filtration through a 0.45 mu m filter, 600 mu l of the collected supernatant was used for reverse-phase HPLC analysis.

The reverse phase HPLC (LC-20AD Liquid Chromatograph system and SPD-20A UV / VIS Detecror; SHIMADZU) analysis was performed using a Columbus 5 urn C18 column (1504.60 mm; PerkinElmer) and a Columbus 5 urn C18 column (1504.60 mm; PerkinElmer) Was maintained at 25 占 폚 with 20% methanol in flowing water at a rate of 1.0 ml / min. Each peak value of the chromatogram was analyzed at 210 and 268 nm. Cipophil and coniferyl alcohol 4-O-glucoside have been identified by comparison with known standards (CAS No.532-29-3 and CAS No.118-34-3, National Institutes for Food and Drug Control, China). Data were analyzed using SHIMADZU LC solution software and quantitative measurements were made based on ultraviolet absorption at 268 nm. The highest numerical value of the data was calculated by comparing it with the external standard calibration curve.

Example  1. Transformant vector and transformant production

Two transgenic vectors were constructed using the 35S promoter over-expressed in all plants and the beta-conglycine promoter that specifically reacted with seeds (Fig. 1). As shown in Fig. 2, the transformants into which the recombinant herpes simplex enzyme UGT72E-3/2 protein coding gene was inserted were transformed into 12 mutants (35S-UGT72E-3/2) and 7 mutants (? -UGT72E-3/2).

Example  2. Confirmation of introduction of inserted gene

For 35S-UGT72E-3/2 T 0 transformants to confirm the switch body and the β-UGT72E-3/2 T 0 transformants inserted gene in transformants was carried out PCR and RT-PCR.

As a result of PCR, insertion of UGT72E-3/2 gene, which is an introduced gene, was confirmed in most transformants (FIG. 3). RT-PCR was performed to confirm the expression and the expression level of UGT72E-3/2 gene. Two of the transformants (35S-UGT72E-3/2) and three (β- UGT72E-3/2) (Fig. 4). At this time, β-tubulin was used as a reference gene, and the difference in the degree of expression could be confirmed separately from the expression of UGT72E-3/2 gene.

Example  3. 35S- UGT72E -3/2 and < UGT72E -3/2 of the transformant Sirin Jin Coniferin  Content analysis

Reversed-phase HPLC analysis of the contents of silginin and conipiperin in the leaves obtained from the two transformants showed that both serine and conipherin, which were not detected in the wild-type light bean (WT) UGT72E-3/2, and-UGT72E-3/2) (Fig. 5).

Attach an electronic file to a sequence list

Claims (10)

Comprising the step of transfecting a soybean plant cell with a recombinant vector comprising a gene encoding a recombinant glycosyltransferase UGT72E-3/2 protein consisting of the amino acid sequence of SEQ ID NO: 2 to over-express the UGT72E-3/2 gene To increase syringin and coniferin content in soybean plants. delete delete Expressing the UGT72E-3/2 gene by transforming a soybean plant cell with a recombinant vector comprising a gene encoding a recombinant glycosyltransferase UGT72E-3/2 protein comprising the amino acid sequence of SEQ ID NO: 2; And
Wherein the syringin and coniferin contents are increased as compared to the wild type, comprising regenerating soybean plants from the transformed soybean plant cells. delete delete A transformed soybean plant having increased syringin and coniferin contents compared to the wild type produced by the method of claim 4. A seed of a transgenic soybean plant according to claim 7. A recombinant vector comprising the gene encoding the recombinant glycosyltransferase UGT72E-3/2 protein consisting of the amino acid sequence of SEQ ID NO: 2 and having increased syringin and coniferin contents compared to the wild type Conversion soybean plants. For increasing syringin and coniferin content in soybean plants containing a recombinant vector comprising the gene encoding the recombinant glycosyltransferase UGT72E-3/2 protein consisting of the amino acid sequence of SEQ ID NO: 2 as an active ingredient Composition.

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