KR20150047787A - Transgenic soybean plant with enhanced tolerance to Soybean mosaic virus and production method thereof - Google Patents

Abstract

The present invention relates to an enhancing method of tolerance in plants to viral disease by soybean mosaic virus, comprising a step of transforming to a recombinant plant RNAi expression vector containing a helper component-proteinase (HC-Pro) gene derived from the soybean mosaic virus, transforming a resistance enhanced plant to the viral disease by soybean mosaic virus, a seed thereof, and the vector to the plants, and inducing gene silencing of the HC-Pro gene.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a soybean transgenic plant having enhanced tolerance to soybean mosaic virus and a method for producing the soybean transgenic plant,

The present invention relates to a soybean transgenic plant having enhanced resistance to soybean mosaic virus and a method for producing the soybean transgenic plant. More particularly, the present invention relates to a soybean mosaic virus-derived helper component-proteinase gene derived from soybean mosaic virus A gene and a seed thereof transformed with a recombinant plant RNAi expression vector of the HC-Pro gene, which promotes resistance to viral diseases caused by soybean mosaic virus, and gene silencing of the HC-Pro gene by transforming the vector into a plant, And a method for promoting tolerance of a plant to viral diseases caused by soybean mosaic virus.

RNA silencing is a virus defense mechanism that can protect against virus infection. Plant viruses have RNA genomes and are known to be targets of RNA silencing inducers and post-transcriptional gene silencing (PTGS). On the other hand, some viruses encode proteins that inhibit RNA silencing. The potyvirus RNA silencing inhibitor, HC-Pro ( Helper - component proteinase is a multifunctional protein that is encoded by plant viruses and is involved in the stages of a viral infection cycle, replication, mediation of aphids, and systemic and viral cell-to-cell movement of viruses. In particular, HC-Pro is identified as an inhibitor of PTGS that blocks accumulation of siRNA.

RNA interference (RNAi) is one of the interesting biotechnological approaches to gene function analysis in plants and is a virus defense mechanism, also known as PTGS. An important key to RNAi is in the production of double-stranded RNA (dsRNA), an initiator of PTGS. The dsRNAs are cleaved into siRNAs (small interference RNAs) of 21-23 nucleotides by an RNase III-like enzyme called dicer. These siRNAs bind to RISC (RNA-induced silencing complex). RISC unravels double-stranded siRNAs, uses siRNA as a guide to bind target mRNAs, and cleaves mRNAs that cause the inhibition of specific gene expression. Therefore, RNAi can be an important tool to knockdown target genes and silence the clear characteristics of plants. As a result, viral disease in plants can be controlled and crops can be protected from viruses and insects.

Soybean mosaic virus ( Soybean mosaic virus , SMV) is the most well-known viral disease in soybeans, belonging to Potyviridae, which has a positive sense single-stranded RNA genome of about 10 kb. Most viral symptoms are found in leaves and seeds of infected beans. Leaves have symptoms such as mosaic pattern, bending and necrosis. In the case of seeds, spots and speckles appear on the seed coat. In particular, infected soybean plants have lost crop yields due to a noticeable reduction in seed count. The transmission of the soybean mosaic virus is not only spread from parent to offspring, but also from the migration of other kinds of aphids.

Soybean (Glycine max (L.) Merr.) Is the world's leading crop, providing the best vegetable oil and protein for food and drinks. Considering soybeans as important crops, transgenic technology has been widely used to improve valuable genetic characteristics in soybeans. Soybean transgenic plants introduced with various useful genes have been developed using Agrobacterium-mediated transformation system based on the cotyledonary-node (CN) method, and recently, half-seed explants ) To improve the system. In addition, the use of additional thiol compound blends and the application of sonication and vacuum treatment resulted in a positive improvement in the transformation efficiency.

The present invention relates to an inhibitor of soybean mosaic virus HC - Pro In order to apply the suppressor HC - Pro gene to the RNAi principle, Agrobacterium - mediated transformation technique was introduced into soybean to produce a transformant. As a result, a soybean transformant having resistance to soybean mosaic virus was developed The researchers focused on research.

Viral resistant crops can also be produced by breeding, but production of specific virus resistant crops using biotechnology rather than breeding has never been attempted. Considering the fact that disease-resistant breeding materials are getting depleted, development of methods other than breeding is very encouraging. Thus, in the present invention, an inhibitor HC-Pro gene of soybean mosaic virus was introduced into the RNAi principle by using a method other than hybridization method using biotechnology, and as a result, a soybean mosaic virus We want to see if the body is produced.

Korean Patent Laid-Open Publication No. 2012-0041608 discloses a recombinant VIGS vector derived from bean macular mosaic virus and its use for the analysis of the function of useful genes in soybean. However, as in the present invention, soybean mosaic virus There has been no report on a transgenic plant and its production method.

DISCLOSURE OF THE INVENTION The present invention has been made in view of the above needs, and it is an object of the present invention to provide a recombinant plant RNAi expression vector which induces gene silencing of the HC-Pro gene, The present inventors have completed the present invention by confirming that they exhibit a significant increase in tolerance compared to wild type plants.

In order to solve the above problems, the present invention provides a recombinant plant RNAi expression vector comprising HC-Pro (helper component-proteinase) gene derived from soybean mosaic virus.

The present invention also provides a method for producing a plant having enhanced resistance to viral diseases caused by soybean mosaic virus using the recombinant plant RNAi expression vector.

In addition, the present invention provides a plant produced by the above method, wherein the soybean mosaic virus is resistant to viral diseases, and a seed thereof.

The present invention also relates to a method for transforming a plant with a recombinant plant RNAi expression vector comprising HC-Pro gene derived from soybean mosaic virus to induce gene silencing of the HC-Pro gene The present invention provides a method for promoting tolerance of a plant to a viral disease caused by a soybean mosaic virus.

The present invention also relates to a plant for the viral disease caused by soybean mosaic virus, which contains, as an active ingredient, a recombinant plant RNAi expression vector containing HC-Pro (helper component-proteinase) gene derived from soybean mosaic virus A composition for enhancing the tolerance of < RTI ID = 0.0 >

According to the present invention, when HC-Pro gene of soybean mosaic virus is introduced into RNAi vector and introduced into soybean using Agrobacterium-mediated transformation technique, soybean transformant which is resistant to soybean mosaic virus is produced .

Fig. 1 is a schematic diagram of a soybean transformation vector { HC- Pro (RNAi)} of the present invention.
Fig. 2 shows the growth process of soybean plants transformed with HC - Pro (RNAi).
Figure 3 shows the results of PCR performed to confirm the inserted gene in the HC-Pro (RNAi) T ₀ transformants. NT: Gwangan bean (wild type), EV: Boll vector, 1-9: Transformant.
FIG. 4 shows the results of Southern blotting performed to identify the inserted gene in the HC-Pro (RNAi) T ₂ transformant. NT: Gwanan bean (wild type), EV: Ball vector, # 1-6: T ₂ transformant.
Fig. 5 shows the test of soybean mosaic virus in a HC-Pro (RNAi) T ₂ transformant in which virus was artificially infected. NT: Manganese bean (wild type), NT (M): Gwangan bean (wild type) infected with virus, EV (M): Viral infected vector, 1-6: Transfected virus, Resistance, mM: mild mosaic symptoms, and m: mosaic symptoms.
FIG. 6 shows Northern blotting results for virus expression analysis in HC-Pro (RNAi) T ₂ transformants in which viruses were artificially infected. NT: Manganese bean (wild type), NT (M): Gwangan bean (wild type) infected with virus, EV (M): viral infected vector, # 1-6: Virus resistance, mM: mild mosaic symptoms, and m: mosaic symptoms.
FIG. 7 shows the results of RT-PCR performed for virus expression analysis in HC-Pro (RNAi) T ₂ transformants in which viruses were artificially infected. NT: Manganese bean (wild type), NT (M): Gwangan bean (wild type) infected with virus, EV (M): viral infected vector, # 1-6: Virus resistance, mM: mild mosaic symptoms, and m: mosaic symptoms.
Figure 8 shows the morphology of T ₃ seeds harvested from HC - Pro (RNAi) T ₂ transformants that were infected with the virus artificially. NT: Manganese bean (wild type), NT (M): Gwangan bean (wild type) infected with virus, EV (M): viral infected vector, # 1-6: Virus resistance, mM: mild mosaic symptoms, and m: mosaic symptoms.
FIG. 9 shows the results of RT-PCR performed for virus expression analysis of T ₃ seeds harvested from HC - Pro (RNAi) T ₂ transformants in which viruses were artificially infected. NT: Manganese bean (wild type), NT (M): Gwangan bean (wild type) infected with virus, EV (M): viral infected vector, # 1-6: Virus resistance, mM: mild mosaic symptoms, and m: mosaic symptoms.

In order to accomplish the object of the present invention, the present invention provides a recombinant plant RNAi expression vector comprising HC-Pro (helper component-proteinase) gene derived from soybean mosaic virus.

The HC-Pro gene refers to a sequence containing a cysteine proteinase domain in the HC-Pro gene, preferably a nucleotide sequence of SEQ ID NO: 1, but is not limited thereto.

The recombinant plant RNAi expression vector may be, for example, the pB7GWIWG2 (I) :: HC-Pro vector described in FIG. 1, 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.

The term "vector" is used to refer to a DNA fragment (s), nucleic acid molecule, which is transferred into a cell. The vector replicates the DNA and can be independently regenerated in the host cell. The term "carrier" is often used interchangeably with "vector ". The term "expression vector" means a recombinant DNA molecule comprising a desired coding sequence and a suitable nucleic acid sequence necessary for expressing a coding sequence operably linked in a particular host organism. Promoters, enhancers, termination signals and polyadenylation signals available in eukaryotic cells are known.

A preferred example of a plant expression vector is a Ti-plasmid vector that is capable of transferring a so-called T-region into a plant cell when it is 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 will preferably comprise 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 plant expression vector of the present invention, the promoter may be CaMV 35S, actin, ubiquitin, pEMU, MAS or histone promoter, but is not limited thereto. The term "promoter " refers to the region of DNA upstream from the structural gene and refers to a DNA molecule to which an RNA polymerase binds to initiate transcription. A "plant promoter" is a promoter capable of initiating transcription in plant cells. A "constitutive promoter" is a promoter that is active under most environmental conditions and developmental conditions or cell differentiation. Constructive promoters may be preferred in the present invention because the choice of transformants can be made by various tissues at various stages. Thus, constitutive promoters do not limit selectivity.

In the plant expression vectors 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. Therefore, the use of a terminator is highly desirable in the context of the present invention.

The present invention also provides a method for producing a plant cell, which comprises transforming a plant cell with a recombinant plant RNAi expression vector comprising a HC-Pro helper component-proteinase gene derived from a soybean mosaic virus; And

And regenerating the transgenic plant from the transformed plant cell. The present invention also provides a method for producing a plant having enhanced resistance to viral diseases caused by soybean mosaic virus.

Transformation of a plant means any method of transferring DNA to a plant. Such transformation methods do not necessarily have a regeneration and / or tissue culture period. In principle, any transformation method can be used to introduce the hybrid DNA according to the present invention into suitable progenitor cells. The method is based on the calcium / polyethylene glycol method for protoplasts (Krens, FA et al., 1982, Nature 296, 7274; Negrutiu I. et al., June 1987, Plant Mol. Biol. 8, 363373) (Shillito RD et al., 1985 Bio / Technol. 3, 10991102), microinjection into plant elements (Crossway A. et al., 1986, Mol. Gen. Genet. 202, 179185) Or RNA coated (Klein et al., 1987, Nature 327, 70), infiltration of plants or Agrobacterium tumefaciens mediated gene transfer by transformation of mature pollen or micropellets ) Virus infection (EP 0 301 316) and the like. A preferred method according to the present invention comprises Agrobacterium mediated DNA delivery. Particularly preferred is the use of so-called binary vector techniques as described in EP A 120 516 and U.S. Pat. No. 4,940,838.

"Plant cell" used for transformation of a plant may be any plant cell. Plant cells are cultured cells, cultured tissues, cultivated or whole plants.

"Plant tissue" refers to a tissue of differentiated or undifferentiated plant, including but not limited to roots, stems, leaves, pollen, seeds, cancer tissues, and various types of cells used for culture, protoplasts, shoots and callus tissue. The plant tissue may be in planta or may be in an organ culture, tissue culture or cell culture.

In addition, the present invention provides a plant produced by the above method, wherein the soybean mosaic virus is resistant to viral diseases, and a seed thereof.

Transgenic plants in the present invention may include whole plants as well as tissues, cells or seeds obtainable therefrom.

Plants transformed with the HC-Pro fusion gene of the present invention have excellent resistance to viral diseases caused by soybean mosaic virus.

The plant is preferably a host plant of soybean mosaic virus, and may be a soybean plant such as pea, kidney bean, and the like, but is not limited thereto.

The present invention also includes a step of transforming a recombinant plant RNAi expression vector comprising the HC-Pro gene consisting of the nucleotide sequence shown in SEQ ID NO: 1 into a plant to induce gene silencing of the HC-Pro gene The present invention provides a method for promoting tolerance of a plant to viral diseases caused by soybean mosaic virus.

As a method for inducing gene silencing of the HC-Pro gene, various methods known in the art can be used. The present invention provides a method of transforming a plant as a gene silencing construct directed toward a plant virus gene so that the plant has resistance to the virus.

The present invention also relates to a plant for the viral disease caused by soybean mosaic virus, which contains, as an active ingredient, a recombinant plant RNAi expression vector containing HC-Pro (helper component-proteinase) gene derived from soybean mosaic virus A composition for enhancing the tolerance of < RTI ID = 0.0 > The composition of the present invention contains a recombinant plant RNAi expression vector containing an HC-Pro (helper component-proteinase) gene derived from soybean mosaic virus as an active ingredient, and the recombinant plant RNAi expression vector is transformed into a plant Thereby promoting tolerance of the plant to viral diseases caused by soybean mosaic virus. The plant may be, but is not limited to, beans.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Experimental Method

1. Inhibitors of soybean mosaic virus HC - Pro Introduced RNAi  Vector production

HC - Pro is a soybean mosaic virus strain G7H gene and was provided by Professor Kim, Kook Hyung of Seoul National University. The cDNA fragment for subcloning was prepared by PCR. The amplified gene was subcloned into pENTR / D-TOPO vector (Invitrogen, USA) to confirm the base sequence. In order to construct the confirmed pENTR- HC - Pro on the final target vector pB7GWIWG2 (I), Gateway LR Clonase ( ^TM ) of Invitrogen Enzyme mix kit was used and transformed by using DH5α of Invitrogen as a competent cell. Built pB7GWIWG2 (I) - HC - Pro The vector (Figure 1) was transformed into Agrobacterium tumefaciens strain EHA105.

2. Soybean transformants and T _One  Seed production

2-1. Seed disinfection and immersion

The seeds were sterilized with hydrochloric acid gas generated by mixing lactose (12 N sodium chloride, 100 mL) and lactose (5 mL of 12N HCl), and incubated in 1% sodium hypochlorite for 10 minutes Secondly, they were rinsed three times with sterilized water every 10 minutes. The sterilized seeds were placed in a 50-mL conical tube, and sterilized water was poured thereinto for 20 hours at room temperature.

2-2. Inoculum ( Agrobacterium tumefaciens ) preparation

For the bean transformation experiment, the pB7GWIWG2 (I) -HC -Pro vector constructed in Agrobacterium tumefaciens strain EHA105 was used and PPT ^R . (100 mg / L of kanamycin, 25 mg / L of rifampicin, 10 g / L of peptone, 5 g / L of NaCl and 5 g / L of yeast extract, 1.5% After incubation at 25 ° C, the resulting single colonies were placed in 10 mL of a liquid YEP medium containing the same antibiotic and stirred at 250 rpm at 25 ° C until the OD ₆₅₀ reached 0.6-0.8. 10 mL of 30% glycerol stock was added to the incubated culture, mixed with 1 mL each in a 1.5 mL tube, rapidly cooled with liquid nitrogen, and stored at -70 ° C. The inoculated Agrobacterium tyumeo Pacific Enschede stock (competent cell) sewn to antibiotics are stored at -70 ℃ in liquid YEP medium 200 mL containing 1 ml the day before into the OD ₆₅₀ until the 0.6 ~ 0.8 at 25 ℃ 250 rpm In an incubator.

On the day of inoculation, 200 mL of liquid YEP medium was divided into 50 mL portions and centrifuged at 3270 g for 10 minutes at 20 캜. Liquid CCM Agrobacterium tyumeo Pacific Enschede pellet in each tube (co-cultivation medium; B5 salt 0.32 g / L, BA 1.67 ㎎ / L, MES 20 mM, GA 3 L-cysteine 3.3 mM, sodium thiosulfate 1.0 mM, DTT 1.0 mM, sucrose 3%, pH 5.4) was added to the solution.

2-3. Inoculation and co-culture

The scalpel was inserted between the cotyledons of the immersed seeds, cut vertically to the hypocotyl, and removed the seed coat. The dorsiflexion was cut at a distance of about 1 cm under the cotyledon, and one side with the embryonic axis was wound about 7 · 8 times with a scalpel (# 11 blade). At this time, 15 mL of concentrate was applied to the surgical scalpel and wound to the target area. Approximately 50 pieces of the section were placed in a 15 mL co-culture / Agrobacterium tumefaciens, sonicated for 20 seconds, and placed in a clean bench in a desiccator (250 mm (width) x 250 mm (length) x 300 mm (height)} and a diaphragm pump (GAST) for 30 seconds (500 mm.Hg) and then inoculated for 30 minutes. Each slice was taken out of the tube and placed on sterile filter paper. After removing the water, 10 sheets of filter paper were placed on the solid CCM (same as liquid, 0.7% agar) and 10 individuals were placed (adaxial portion was downward }. After sealing with Micropore, the cells were incubated for 5 days at 25 ° C for 18 hours.

2-4. Wash and induce shoots

After 5 days of co-cultivation, the cells were simply washed for 10 minutes in liquid 1/2 SIM (shoot induction medium) for sterilization. Each of the slices was placed on a filter paper and the water was removed. Then, SI-1 (shoot induction medium: B5 salt 3.2 g / L, BA 1.67 mg / L, MES 3 mM, agar 0.8%, sucrose 3% 6 cells per plate were fixed to the culture medium and the part to be regenerated was 30 ° or less. The cells were incubated at 37 ° C for 30 min. Of the total length of the flap. Each plate was sealed with a micropore and incubated at 25 ° C for 18 hours.

After 2 weeks, the shoots were sutured to SI-2 (SI-①, DL-phosphinotricin 10 mg / L, pH 5.6) containing 10 ㎎ / L of the antibiotic PPT as the starting antibiotic. The remaining portion was cut off and the adaxial portion was faced downward.

2-5. Shincho Kidney

After 2 weeks, the browning / shoal pad was cut with a surgical scalpel (# 15 blade) and SEM (shoot elongation medium; MS salt 4.4 g / L, MES 3 mM , GA ₃ 0.5 mg / L, asparagine 50 mg / L, pyroglutamic acid 100 mg / L, IAA 0.1 mg / L, zitin 1 mg / L, sucrose 3%, agar 0.8% , Vancomycin 50 mg / L, ticarcillin 100 mg / L, DL-phosphinotricin 5 mg / L, pH 5.6). Every 2 weeks, it was transferred to a new SEM, and the brow portions of the shoots were struck by the upper surface of a surgical scalpel (# 15 blade), and the shoot pad was gradually shaved to absorb the medium well.

2-6. Root Formation, Circulation Process and PPT Leaf Painting

(Root induction medium; MS salt 4.4 g / L, MES 3 mM, sucrose 3%) was cut with a surgical scalpel (# 11 blade) L, 50 mg / L of Tacarcine, 25 mg / L of asparagine, 25 mg / L of pyroglutamic acid, pH 5.6). At this time, the bottom of the cut and elongated shoots was immersed in 1 mg / mL IBA for 3 minutes, taken out, and placed in a test tube containing RM.

When the roots grow sufficiently, wash the medium with the third distilled water and plant it in a small pot (6 cm x 6 cm x 5.6 cm) mixed with 2: 1 mixture of soil (Bio-Phrag 2) and vermiculite (magenta box Magenta box). After about 10 days, the leaf surface was subjected to leaf painting with 100 ㎎ / L DL-phosphinothricin.

2-7. T ₁ seed production

When the plant was sufficiently grown, it was transferred to a larger pot and about 10 holes were made in the clear plastic cover. After 10 days, the plants were treated with Bastar (BAYER, 53 mg / L). As a result, non - transgenic plants (Kwangan soybean, NT) were sensitive and died. On the other hand, the transformants showed no change and showed resistance. 9 bean transformants showing resistance were transferred to the greenhouse and T ₁ The seeds were harvested.

3. Analysis of gene transfer

3-1. PCR and Southern blotting analysis

1 g of the leaves of the transgenic plants were quantified, frozen with liquid nitrogen, frozen leaves were put into a mortar, and the genomic DNA was extracted using the CTAB method. PCR was performed using the transgene and Bar sequence, the first antibiotic gene. Between the HC- Pro (forward primer, 5'-GCAGCGGCAATAAGCCAGTCAATT-3 ': SEQ ID NO: 2) and the intron (reverse primer, 5'-TGATGGCCATAGGGGTTTAGATGC-3': SEQ ID NO: 3) in order to confirm the introduction of the HC- DNA between the DNA and intron (forward primer, 5'-GCATCTAAACCCCTATGGCCATCA-3 ': SEQ ID NO: 4) and HC- Pro (reverse primer, 5'-GCAGCGGCAATAAGCCAGTCAATT-3': SEQ ID NO: 5) was amplified. The primers used for the introduction of Bar, the first antibiotic gene, were as follows. Bar forward primer (5'-TGGCTGGTGGCAGGATATATTGTG-3 ': SEQ ID NO: 6) and Bar reverse primer (5'-AGACAAGCACGGTCAACTTCCGTA-3': SEQ ID NO: 7). The left border was amplified by PCR to see if the insertion of T-DNA was complete. The left border portion used the left border forward primer (5'-TGGCTGGTGGCAGGATATATTGTG-3 ': SEQ ID NO: 8) and the Bar reverse primer (5'-AGACAAGCACGGTCAACTTCCGTA-3': SEQ ID NO: 9).

To analyze Southern blotting, approximately 10 μg of genomic DNA per line was digested with Hind III for overnight enzymatic digestion with Ganban bean and each transformant. The digested DNA was electrophoresed on 1% agarose gel. The separated DNA was transferred to a nylon membrane (Hybond-N ⁺ , Amersham, UK) using capillary action. Hybridization, washing and detection were performed using DIG (digoxigenin) -labeled DNA probes and a chemiluminescent system (Roche, Germany). The DIG-labeled DNA probes were labeled with Bar Was prepared by PCR amplification using a forward primer (5'-AACTTCCGTACCGAGCCGCA-3 ': SEQ ID NO: 10) and a Bar reverse primer (5'-TCGTAGGCGTTGCGTGCCTT-3': SEQ ID NO: 11).

4. Virus inoculation of transformant and Symptom  analysis

4-1. Virus inoculation of transformant

When harvested soybean seeds were sown, they were inoculated with soybean mosaic virus strain G5 after germination one week later. A sample of soybean leaves preliminarily found in Muller's bowl was ground with buffer (0.1M phosphate buffer, pH 7.2) to prepare juice, and carborundum (Nalalai tesque, 600mesh) was evenly distributed on the whole leaf of the beans to be assayed. The prepared juice was put on a finger with rubber gloves and rubbed the surface of the bean leaves with carborundum. After the inoculation, the juice was sufficiently washed and rinsed with water to remove iron powder (Zheng et al., 2005, Crop Prot., Vol.24, 49-56; Zheng et al., 2006, J. Hered. , Vol. 97 (5), 429-437). Viruses were analyzed at weekly intervals until 4th week. In the case of viral disease, transformants resistant to viruses were classified as resistance (R), weak mosaic symptoms as mild mosaic, and strong mosaic symptoms as mosaic (M, mosaic). The virus symptoms of harvested seeds were classified as follows. The clean seeds were classified as resistance (R), mild mosaic (mild mosaic) for light seed coat, and mosaic (mosaic) for dark seed coat stain.

4-2. Virus expression analysis

To determine the expression of viral RNA, RNA was extracted from the transformants inoculated with the virus and harvested seeds, and Northern blotting and RT-PCR were performed. For transformants inoculated with the virus, RNA was isolated using a plant RNA purification solution (Invitrogen). For the seeds, the seeds were finely ground with 0.1 M Tris-HCl (pH 9.0) cooled to 4 ° C by 40 times the sample weight, and the RNA was isolated using a plant RNA purification solution (Invitrogen).

For the northern blotting analysis of the virus-inoculated transformants, 20 μg of the RNA isolated in the above was electrophoresed on a 1.2% (w / v) denaturing formaldehyde agarose gel to prepare a nylon membrane (Hybond-N ⁺ , Amersham , UK). Hybridization, washing, and detection were performed using a DIG (digoxigenin) -labeled DNA probe and a chemiluminescence system (Roche, Germany). The DIG-labeled probe was amplified by PCR amplification using HC - Pro forward primer (5'-CACGAAGATGGTAAGGGATG-3 ': SEQ ID NO: 12) and HC - Pro reverse primer (5'-GGTACCCAACTGTCAAGGAT-3' Ready.

The RNA isolated from the seeds was subjected to RT-PCR using Maxime RT-PCR PreMix (iNtRON, KOREA). Transgene in HC - to determine the transcription levels of HC Pro - Pro forward primer (5'-CACGAAGATGGTAAGGGATG-3 ': SEQ ID NO: 12) and HC - Pro reverse primer (5'-GGTACCCAACTGTCAAGGAT-3': SEQ ID NO: 13) Were used for RT-PCR.

Example  1: Transformant vector and transformant production

SMV ( Soybean mosaic virus ) Transformation vector was prepared using the HC-Pro gene derived from strain G7H and RNAi (Fig. 1). As shown in Fig. 2, the transformants were subjected to plant regeneration to produce 9 transgenic plants.

Example  2: for confirmation of gene transfection of transformants PCR  analysis

According to the method described above, HC - Pro (RNAi) T ₀ DNA was isolated from the transformants and PCR was carried out. As a result, HC - Pro gene, Bar gene, the first antibiotic gene, and the left border were confirmed (FIG. 3). Southern blotting was performed to determine the number of copies in the T ₂ transformants, and one or two low copy numbers were found in most transformants (FIG. 4).

Example  3: Virus of soybean transformants infected with virus Symptom  Expression analysis of black and viral genes

In order to observe the virus infection symptoms of HC - Pro (RNAi) T ₂ transformant, SMV (soybean mosaic virus) strain G5 was inoculated at the beginning of germination one week after germination. In the case of viral disease, the transformants resistant to the virus were classified as resistance (R), weak mosaic symptoms as mild mosaic, and strong mosaic symptoms as mosaic (mosaic). In the leaves infected with virus, light and dark green mosaic pattern occurred and leaf bending was observed. Transformant lines 2, 5, and 6 showed no virus symptoms and showed resistance (R). On the other hand, transformant lines 1, 3 and 4 showed mild mosaic (mM) or mosaic (M) symptoms (FIG. 5). Northern blotting was performed to analyze the expression of the virus HC-Pro gene according to the symptoms of viral infection. As a result, viral RNA was not expressed in the resistant lines 2, 5 and 6. On the other hand, viruses were strongly expressed in the senile lines 1, 3 and 4 (Fig. 6). In addition, gene expression analysis of HC-Pro, CP (coat protein) and CI protein (CI), which are proteins encoded by potyvirus RNA, was performed by reverse transcriptase PCR analysis and found to be similar to northern blotting (Fig. 7).

Example  4: Morphology and viral expression analysis of seeds harvested from virus-infected soybean transformants

To determine whether the virus symptoms of the virus-infected plants affected the seed morphology, we examined whether the virus was caused by harvested T ₃ seeds. The virus symptoms of harvested seeds were classified as follows. The clean seeds were classified as resistance (R), mild mosaic (mild mosaic) for light seed coat, and mosaic (mosaic) for dark seed coat stain. As a result, the T ₃ seeds of the resistant lines 2, 5 and 6 were all clean and resistant and no seed coat mottling was produced. When compared with virus-resistant soybean seeds, brown or black seed coat staining, which is a virus symptom, was observed in the T ₃ seeds of the senile lines 1, 3 and 4 (FIG. 8). RT-PCR was performed to analyze the expression of HC-Pro, CP, and CI protein genes encoded by potyvirus RNA from these seeds, and the result showed a pattern similar to the analysis of viral expression in leaves (FIG. 9). Resistance lines 2, 5 and 6 were not in T ₃ seed of the viral RNA expression in T ₃ seeds Susceptible lines 1, 3 and 4, it was confirmed that the virus was developed. These results indicate that the seeds of Lee Byeongseong line were greatly influenced by virus-infected plants. In other words, it was confirmed that the virus was correctly transferred from the T ₂ plant to the T ₃ seed. Viruses were not analyzed in virus-resistant transformants, and resistance persisted throughout the generation.

<110> Dong-A University Research Foundation For Industry-Academy Cooperation <120> Transgenic soybean plant with enhanced tolerance to Soybean          mosaic virus and production method thereof <130> PN13331 <160> 13 <170> Kopatentin 2.0 <210> 1 <211> 305 <212> DNA <213> Soybean mosaic virus <400> 1 atgcctccta atgtggagaa tcatgaatgc accattgatt tcacaaatga acaatgtggt 60 gaactggcag cggcaataag ccagtcaatt tttccagtta agaaactatc atgcaagcaa 120 tgtcggcagc acattaagca cctcagttgg aggagtataa acaattcctc ttggctcata 180 tgggctgcca tgggaccgaa tgggaaactt tccaagaaat tgacggcatg aagtatgtga 240 agagagtgat tgagacatca actgcggaaa acgcaagtct gcagacatca ctggagattg 300 tgcgt 305 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 gcagcggcaa taagccagtc aatt 24 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 tgatggccat aggggtttag atgc 24 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 gcatctaaac ccctatggcc atca 24 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 gcagcggcaa taagccagtc aatt 24 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 tggctggtgg caggatatat tgtg 24 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 agacaagcac ggtcaacttc cgta 24 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 tggctggtgg caggatatat tgtg 24 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 agacaagcac ggtcaacttc cgta 24 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 aacttccgta ccgagccgca 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 tcgtaggcgt tgcgtgcctt 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 cacgaagatg gtaagggatg 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 ggtacccaac tgtcaaggat 20

Claims (11)

A recombinant plant RNAi expression vector comprising HC-Pro (helper component-proteinase) gene derived from soybean mosaic virus. 2. The recombinant plant RNAi expression vector according to claim 1, wherein the HC-Pro gene comprises the nucleotide sequence of SEQ ID NO: 1. 2. The recombinant plant RNAi expression vector according to claim 1, which is the pB7GWIWG2 (I) :: HC-Pro vector described in Fig. Transforming a plant cell with a recombinant plant RNAi expression vector according to any one of claims 1 to 3; And
And regenerating a transgenic plant from the transformed plant cell, wherein the soybean mosaic virus is resistant to viral diseases. A plant produced by the method of claim 4, wherein the soybean mosaic virus promotes resistance to viral diseases. 6. The plant according to claim 5, wherein the plant is a soybean. A plant seed according to claim 5. A method for inducing gene silencing of a HC-Pro gene by transforming a recombinant plant RNAi expression vector containing HC-Pro gene derived from soybean mosaic virus into a plant to induce gene silencing of the HC- &Lt; / RTI &gt; to increase the tolerance of the plant to viral diseases. 9. The method of claim 8, wherein the plant is soy. A method for promoting tolerance of a plant to a viral disease caused by a soybean mosaic virus, which comprises, as an active ingredient, a recombinant plant RNAi expression vector comprising a HC-Pro (helper component-proteinase) gene derived from a soybean mosaic virus / RTI &gt; 11. The composition of claim 10, wherein the plant is a soy.

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