KR100350214B1 - Method for generating transgenic plants having multi-virus resistance by expressing ds RNA-dependent protein kinase in the plants - Google Patents

Abstract

The present invention relates to a method of imparting complex resistance to various types of viruses to plants, and more particularly, protein kinase (ds RNA-) activated by double-stranded RNA derived from animals or yeast, preferably humans. The present invention provides a method for producing a plant having complex resistance to various plant viruses having ds RNA as a replication intermediate by transforming the plant with a dependent protein kinase (hereinafter referred to as PKR) gene. In addition, since the method of the present invention introduces a gene of an animal or a yeast into a plant, it may block the risk of emergence of a modified virus due to recombination that may occur when a gene derived from a virus is introduced, and impart complex virus resistance to the plant. Unlike conventional methods for doing so, it has the advantage of not causing morphological and physiological side effects in plants, and thus can be usefully used to prepare plants that are stably resistant to various viruses while maximizing the function of useful plants. have.

Description

Method for generating transgenic plants having multi-virus resistance by expressing ds RNA-dependent protein kinase in the plants}

The present invention relates to a method of imparting complex resistance to various kinds of viruses to plants, and more particularly to transforming plants with PKR genes derived from animals or yeasts, preferably humans, to induce translational initiation factor eIF2α. The present invention relates to a method for producing a plant that exhibits complex resistance to various plant viruses having ds RNA as a replication intermediate through a mechanism of activating the virus to inhibit replication of the virus.

Most agriculturally important plants are infected by plant viruses or viroids, resulting in a significant decrease in yield as well as a significant drop in economic value. Until now, many studies have been conducted to prevent or treat diseases caused by plant viruses, but no satisfactory method has been developed. For example, a method has been developed to impart viral resistance to plants by stimulating the host plant to maintain an antiviral state by expressing some constitutive genes of the virus in the plant. At this time, a virus-derived gene mainly used is a coat protein gene (Abel et al ., Science 232 : 738-743, 1986; Hemeway et al ., EMBO J. 7 : 1273-1280, 1988; Beachy et. al ., Annu. Rev. Phytopathol. 28 : 451-474, 1990), or nonstructural genes, such as the 54 kDa replicase gene (Golemboski et al ., Proc. Natl. Acad. Sci). USA 87 : 6311-6315, 1990), satellite RNAs (Gerlach et al ., Nature 328 : 790-802, 1987; Harrison et al ., Nature 328: 802-805, 1987) or untranslated sites (untranslated sequences) (Lindbo et al ., virology 189 : 725-733 1992). However, a method of imparting viral resistance to a plant by expressing a gene derived from a virus in a plant as described above generally has a problem of imparting resistance only to a single type of the virus or some viruses closely related thereto.

In addition, such as the mutant virus lacking the assembly function of viroid or encapsulated protein, which is a pathogen consisting of only bare stranded RNA of about 240-380 nucleotides in length, externally used virus as described above. There is a problem in that resistance providing method using a protein gene is difficult to be applied.

In actual crop cultivation, many major food crops are infected by several types of related or unrelated viruses. Therefore, in order to effectively prevent and treat viral diseases, it is urgently required to give a wide range of viral resistance to plants. Some methods have been developed to show the inhibitory effect against various plant viruses, such as ribosomal inactivating protein (RIP) (Lodge et al ., Proc. Natl. Acad. Sci. USA 90 : 7089-7093, 1993) or S-adenosylhomocystein hydolase antisense RNA (Masuta et al ., Proc. Natl. Acad. Sci. USA 92 : 6117-6121, 1995 ) Is expressed in plants. However, the transgenic plants expressing these sequences have a problem of showing abnormal traits such as spots on the leaves, growth inhibition, loss of fertility, and the like.

In vertebrates, interferon acts as the host's primary defense against viral infections. Interferon is synthesized by double-stranded RNA produced during most viral infections, and secreted interferon binds to specific receptors in cells to induce antiviral conditions. One of the enzymes induced by such interferon is PKR, and it is known that PKR is involved in translation control mechanism by phosphorylation of elF2α, an alpha subunit of translation initiation factor, in eukaryotes other than plants.

In other words, when PKR binds to ds RNA and activates itself, it phosphorylates itself and phosphorylates the alpha subunit of translation initiation factor, elF2α. Phosphorylated elF2α prevents interaction with eIF-2B, a GDP / GTP exchange factor. do. This mechanism inhibits the replication of the virus, and eventually PKR confers viral resistance to the host (Meurs et al ., Cell 62 : 379-390, 1990).

It is well known that the regulation of the translational process by phosphorylation of elF2α plays an important role in animals and yeast, but it is still unknown whether this mechanism works in plants or whether PKR can play a similar role in these systems. As far as not clear. However, Langland et al ., Plant Physiology 108 : 1259-1267, 1996 show that in vitro experiments, PKR activated by ds RNA can inhibit protein synthesis by phosphorylating plant elF2α. Gil J. et al ., Biochemistry 39 (25): 7521-7530, 2000, reported that phosphorylation of elF2α regulates protein synthesis in plants using in vivo wheat elF2α. It has been shown to play a role in.

Most plant viruses produce ds RNA as an intermediate when replicated after infection as an ss RNA virus. Therefore, if the PKR gene is correctly expressed in plants and can be activated by ds RNA, which is a replication intermediate of plant viral RNA, this would be an effective way to give plants resistance to various viruses.

Therefore, the present inventors have conducted research to develop a method capable of imparting complex virus resistance to plants while having fewer side effects on plant growth or expression of other traits, and thus, animals or yeasts, which are not derived from viruses, preferably Introducing the human PKR gene into a plant can provide a transgenic plant with complex resistance to various viruses while minimizing the deleterious effects caused by the expression of foreign genes. The present invention has been completed by providing a foreign gene expression system.

Accordingly, an object of the present invention is to provide a method for producing a plant having complex resistance to various viruses and a transformed plant produced by the method.

It is also an object of the present invention to provide an effective plant expression system that can minimize the harmful effects of growth inhibition, morphological changes, loss of fertility due to foreign gene expression in addition to complex viral resistance.

1 is a restriction map showing the structure of the recombinant vector pBCB-PKR prepared by inserting the PKR gene into the pOSTECH94 expression vector,

RB: right border LB: left border

Pbcb: arabidopsis promoter of the bcb1 gene

T7-5 ': transcription termination sequence

2 is a gel photograph showing the results of Southern blot analysis of plants transformed with pBCB-PKR,

3 is a gel photograph showing the results of Northern blot analysis of plants transformed with pBCB-PKR,

C: control group 14: transformation series # 14

I: Extract of leaf inoculated with virus U: Upper leaf extract of inoculated part

Figure 4 is a photograph showing whether or not showing the resistance to the virus 14 days after inoculation of CMV, TEV and PVY virus in tobacco transformed with the PKR gene, respectively,

B, D, E: control group transformed with pOSTECH94

A, C, F: Plant family transformed with PKR gene # 14

Top: plants inoculated with PVY

Discontinued: Plants inoculated with CMV

Bottom: Plant inoculated with TEV

5 is a graph showing the results of virus titers measured by ELISA from leaves inoculated 24 and 48 hours after CMV inoculation,

Gray Bars: SamsunNN

Black Bar: PKR Transformant

Figure 6 is a graph showing the results of measuring the systemic virus proliferation through ELISA 15 days after inoculation of the virus.

mock: negative control plant

PKR14-1: A transformant that does not show any visible viral infection symptoms

PKR14-2: Transformant Showing Symptoms of Viral Infection

SNN: Control group showing symptoms of viral infection

7 is to investigate the SDS-PAGE results (A) of the total protein extracted after 24 hours after wounding tobacco leaves not transfected or not with the PKR gene and the degree of phosphorylation of PKR activated by ds RNA. It is a self-radiography picture showing the result (B) of 10% SDS-PAGE after reacting at 30 ° C. for 10 minutes by adding a reaction solution containing [γ- ³² P] ATP.

In order to achieve the above object, the present invention provides a method for imparting complex virus resistance to a plant by transforming the plant with a recombinant vector for plant transformation comprising a gene encoding PKR.

The present invention also provides an effective plant expression system for expressing the PKR gene in plants.

In the present invention, the term "combined virus resistance" is a term used to distinguish a known virus from which a gene derived from a virus is introduced into a plant to resist the virus of origin or a virus closely related thereto. Or resistance to not only some viruses closely related thereto but also to viruses belonging to various groups that are less relevant. Specifically, the present invention exhibits resistance to many kinds of plant viruses having ds RNA as a replication intermediate.

Hereinafter, the present invention will be described in detail.

In the present invention, the PKR gene may be derived from an animal or yeast, and in particular, it is preferable to use the PKR gene derived from humans. For example GenBank Accession No. U50648 (Charles E. Samuel, Genomics 36 (1): 197-201, 1996), GenBank Accession No. AF167472 (Xu, Z. Et al ., J. Interferon Cytokine Res. 18 (8): 609-616, 1998) and the like, and in a preferred embodiment of the present invention, accession number NM_02759 (Meurs, E, et al., Cell 62 (2): 379-390, 1990) may be used as the PKR gene . It was used for the preparation of transgenic plants having complex virus resistance.

In the present invention, an Arabidopsis blue copper binding protein promoter (BCB promoter) which rapidly activates transcription by wound or viral infection as an example of a preferred recombinant vector for expressing the PKR gene in a plant and binds to the promoter Provided is a plant transformation vector comprising a PKR gene. The BCB promoter rapidly activates transcription of a gene that is regulated by the promoter linked downstream when a wound is caused by a viral infection, and thus is useful for the purpose of imparting resistance to viral infection, as in the present invention. Can be used.

In a preferred embodiment of the present invention, pBCB-PKR (Accession Number: KCTC 0823BP) was used as a recombinant vector for expressing the PKR gene in plants. The recombinant vector pBCB-PKR includes a BCB promoter, a human PKR gene, and a T7-5 'transcription termination sequence in sequence between the right and left borders of the T-DNA of the binary vector. there comprises a kanamycin resistance gene as a selective marker gene, and the detailed structure is disclosed in FIG.

By constructing a plant's foreign gene expression system using the BCB promoter, not only the effective expression of the foreign gene can be achieved, but also the harmful effects caused by the expression can be minimized.

Plants to which complex virus resistance can be imparted by the method of the present invention may include all useful plants such as food crops, vegetable crops, special crops, fruit trees, flowers and feed crops.

In particular, food crops include rice, wheat, barley, corn, soybeans, potatoes, wheat, red beans, oats, sorghum; Vegetable crops include arabidopsis, Chinese cabbage, radish, red pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin, green onion, onion, carrot; Special crops include ginseng, tobacco, cotton, sesame, sugar cane, sugar beet, perilla, peanut, rapeseed; Fruit trees include apple trees, pears, jujube trees, peaches, pears, grapes, citrus fruits, persimmons, plums, apricots, bananas; Flowers include roses, gladiolus, gerberas, carnations, chrysanthemums, lilies, tulips; Forage crops are preferably selected from the group comprising lygras, red clover, orchardgrass, alphaalpha, tolsquescue, perennial lysgrass and the like.

According to the method of the present invention which provides complex resistance to a virus by introducing the PKR gene into a plant, a cucumber mosaic virus (CMV), forty, which is a cucumovirus group, which is a representative virus which causes severe damage to agriculture and economics Plants exhibiting resistance to various plant virus groups including tobacco etching virus (TEV), potato virus Y (PVY), and the like belonging to the virus group can be prepared. That is, the PKR protein is autophosphorylated when it is activated by binding to ds RNA, which is a replication intermediate of a virus, and inactivates it by phosphorylating eIF2α of eukaryotic cells, thereby inhibiting protein synthesis and viral replication. It is possible to exhibit resistance to various plant viruses having as a replication intermediate.

In addition, the present invention provides a recombinant vector pBCB-PKR (Accession No .: KCTC 0823BP) for plant expression for imparting complex virus resistance to plants.

The method for producing a plant transformed with the PKR gene may be performed according to a known method. That is, the recombinant plasmid expressing the PKR gene can be prepared using a known plant expression vector as a base vector, and does not include a general binary vector, a cointegration vector, or a T-DNA site. General vectors designed to be expressed in plants can be used.

Among these, binary vectors include a left border and rightborder of about 25 bp which are involved in infection of foreign genes in T-DNA for plant transformation, and are expressed in plants therein. A vector comprising a promoter site and a polyadenylation signal sequence can be used. The binary vector additionally includes a selection marker gene such as kanamycin resistance gene.As a marker gene for selection of transgenic plants, in addition to antibiotic resistance gene, herbicide resistance gene, metabolism related gene, luciferase, physical Genes related to characteristics, ??-glucuronidase (GUS) or ??-galactosidase (GAL) gene, etc. may be used.

When using a binary vector or a coin integration vector, Agrobacterium ( Agrobacterium- mediated transformation) is used as a strain for transformation to introduce the recombinant vector into a plant ( Agrobacterium tumafaciens) Agrobacterium tumefaciens ) or Agrobacterium rhizogenes can be used.

In addition, when a vector containing no T-DNA site is used, electroporation, microparticle bombardment, polyethylene glycol-mediated uptake, etc. are used to introduce the recombinant plasmid into the plant. Can be.

In a preferred embodiment of the present invention, a recombinant vector pOSTECH94 for plant transformation was prepared by inserting the PKR gene into a binary vector pGA482 by connecting with a BCB promoter, introducing it into Agrobacterium tumefaciens, and then using leaf fragment transformation. Tobacco was transformed and transformants were selected in kanamycin containing medium. It was confirmed by Southern blot analysis that the PKR gene was correctly inserted into the transformant, and as a result, the transgenic plant contained a 2.4 kb gene segment corresponding to the full length sequence of the PKR gene. On the other hand, in order to confirm that the PKR gene is effectively expressed from the transgenic plant, the virus was inoculated to induce the activation of the BCB promoter, and Northern blot analysis was performed. Of course, it was confirmed that the transcript of the PKR gene was expressed at high concentrations throughout the whole plant.

In addition, extracts of plants infected with CMV (cucumovirus group, DSEM PV-0187), TEV (tobacco etching virus, DSMZ PV-0308), and PVY (potyvirus group), respectively, to determine whether the transgenic plants exhibit virus resistance. After dilution, 30 and 50-fold dilution, respectively, the results showed that viral infection symptoms were reduced, and most transgenic plants showed no viral infection symptoms or significantly delayed the time of onset of symptoms.

On the other hand, as a result of analyzing the virus titer by ELISA, the antigen concentration of the control group and the transformant was comparable after 24 hours after the virus inoculation, and after 48 hours, the virus titer of the transformant was significantly decreased compared to the control group. It was. Therefore, it can be seen that the PKR gene effectively inhibits virus replication and / or proliferation and confers substantial viral resistance to plants.

As described above, the viral resistance is based on the introduced PKR gene, and in order to confirm whether the introduced human PKR gene functions properly in plants, another embodiment of the present invention is a phosphorylation assay for measuring changes in the degree of phosphorylation of protein levels ( phosphorylation assay) was performed. As a result, in PKR transformants, the amount of ³² P introduced into p68 was more than two times higher than that of the control group. From this fact, the PKR gene introduced into plants was normally expressed in the transgenic plants and their expression was increased. It can be seen that the phosphorylation level of the protein in the plant is increased.

In transgenic studies, it is very important that the foreign genes introduced do not cause other side effects. In the present invention, plants transformed with the PKR gene were not observed abnormal morphological problems, and showed no effect on growth. In this regard, known methods for imparting resistance to a wide variety of viruses in plants (Lodge et al ., Proc. Natl. Acad. Sci . USA 90 : 7089-7093, 1993; Masuta et al ., Proc. Natl. Acad. Sci . USA 92 : 6117-6121, 1995), it can be seen that the method of the present invention is very effective.

In addition, the method of the present invention for producing a virus resistant plant by introducing a gene not derived from a virus has another advantage. In other words, when a virus protein or antisense RNA is introduced into a plant, there is a risk of recombination due to sequence homology between the introduced gene and the infected virus genome, which is a more severe and modified host than the wild type virus. May result in the emergence of recombinant viruses with a range (Greene et al ., Science 263 : 1423-1425, 1994; Wintermantel et al ., Virology 223 : 156-164, 1994). In the present invention, this risk can be significantly reduced by using genes derived from humans.

Summarizing the above, it can be seen that the transgenic plant expressing the human PKR gene has high resistance to various viruses without showing any external abnormalities. Unlike the existing researches, ds RNA can be widely applied to many important food crops because of its resistance to RNA viruses.

The present invention will be described in more detail based on the following examples.

However, the following examples illustrate the present invention, and the scope of the present invention is not limited thereto.

Example 1 Preparation of Chimeric Gene and Production of Transgenic Plant

Arabidopsis blue copper binding protein gene ( bcb ) promoter (Van Gysel et al ., Gene 136 : 79-85, 1993) was used to specifically induce rapid and high levels of gene expression. A plant transformation vector containing the PKR gene was produced.

First, the binary vector pGA482 (An et al ., Plant Molecular Biology Manual A3: 1-19, 1988) was treated with restriction enzymes Bgl II and Eco RI, followed by the T7-5 'transcription termination sequence (Rosenberg et al. al ., Gene 56 (1): 125-135, 1987). A 1.5 kb Hin dIII- Sma I fragment containing the Arabidopsis bcb promoter (-1294- + 247, Van Gysel et al ., Gene 136 : 79-85, 1993) was inserted into the Hin dIII- Hpa I fragment of the vector. POSTECH94 was produced. Next, recombinant plasmids containing PKR full-length cDNA (Meurs et al ., Cell 62 : 379-390, 1990) were cleaved with Cla I and Bam HI to separate the PKR cDNA. The cleaved DNA fragment was inserted into Cla I- Bgl II of pOSTECH94, and the produced recombinant vector was named pBCB-PKR.

The recombinant vector pBCB-PKR was deposited on July 3, 2000 to the Biotechnology Research Institute Gene Bank (Accession Number: KCTC 0823 BP).

The introduced structure of the prepared vectors are shown in Figure 1, using an electroporation method (electroporation) of the pBCB-PKR in Agrobacterium tumefaciens (Agrobacterium tumefaciens), and then leaf segment transfection method (leaf disc transformation method (Horsh et al ., Science 227 : 1229-1231, 1985) was transformed into tobacco ( Nicotiana tabaccum cv. Samsun NN). Transformed tobacco was selected in MS (Murasighe & Skoog medium) medium containing kanamycin (100 μg / ml).

As a result of self-pollination of mature generation 0 transformants (R ₀ ), all first generation transformants (R ₁ ) produced seeds. First generation seedlings with kanamycin resistance were grown in a greenhouse and analyzed for the insertion of transformed genes and the presence of virus resistance.

Example 2 Southern and Northern Blot Analysis

1) Southern blotting

First, genomic DNA was isolated according to the method of Delaporta et al ., Plant Mol. Biol. Rep. 1 : 19-21, 1983 to confirm that the PKR gene was correctly inserted into the transgenic plant. 10 μg of DNA was treated with restriction enzyme Hin dIII and Pst I, isolated by 0.7% agarose gel electrophoresis, and then transferred to nylon membrane (ICN) using capillary transfer (Park et al . , Plant Mol. Biol. 26 : 1725-1735, 1994). After washing for 5 minutes with 6 × SCC to remove excess agarose was irradiated with ultraviolet (254nm, 0.18 J / Sqcm ² ) to fix the DNA to the nylon membrane.

Hybridization reactions were performed using PKR clones labeled [ν- ³² P] as probes. Hybridization solution (0.5M sodium phosphate, pH 7.2, 7% SDS, 1% BSA, 1mM EDTA) was added to the nylon-bound nylon membrane and reacted at 63 ° C. for 16 hours. The next day, the nylon membrane was washed at 45 ° C. for 10 minutes in 2X SSC, 0.1% SDS, and then washed at 42 ° C. for 15 minutes in 0.2X SSC, 0.1% SDS, then covered with a wrap and put on an X-ray film at -70 ° C. Photosensitive. As a result, it was confirmed that the transformed plant contained a 2.4 kb gene segment corresponding to the full length sequence of the PKR gene (see FIG. 2 ).

2) Northern blotting

Total RNA was isolated from leaf tissues using the RNeasy kit (Quiagen) and then separated using a 1.3% agarose / formaldehyde gel and transferred to nylon membrane. Blots transferred to the nylon membrane were subjected to prehybridization and hybridization according to the method of Park et al ., Plant Mol. Biol. 26 : 1725-1735, 1994.

Transcript levels of the PKR gene in transgenic plants were induced at high concentrations within 24 hours after inoculation of the virus (see FIG. 3 ). In addition, PKR transcripts were expressed throughout the body in similar degree and expression patterns as in virus infected leaves. Since resistance must be expressed systemically as well as the site of infection, such properties are very important for introducing viral resistance genes. RNA extracted from control tobacco did not hybridize with the PKR gene probe.

Example 3 Virus Resistance Analysis

In order to confirm whether the transformed plants prepared in Example 1 exhibit resistance to various plant viruses, viruses belonging to different classification groups were inoculated into the transformed plants. First, the transformed plants were grown in an insect-proof greenhouse having a temperature controlled at 23 ° C. The virus was then inoculated against 4-6 leafed transgenic and control plants (Lim et al ., Mol . Cells 7 : 313-319, 1997).

Inoculated viruses were 15 times for CMV and 15 times for CMV (cucumovirus group, DSMZ PV-0187, Germany), TEV (tobacco etching virus, DSMZ PV-0308), and PVY (potyvirus group). 30 times, 50 times for PVY Diluted to use.

Sixteen first-generation transgenic plants were tested for viral resistance. First, the first-generation seedlings transformed from the kanamycin culture plate were selected, and 20 individuals of each first-generation plant family exhibiting kanamycin resistance were selected. After cultivating in soil, 4-6 leaves were inoculated with CMV, which shows typical systemic symptoms. As a control, CMV was inoculated into a plant transformed with the plant expression vector pOSTECH94 that does not contain the PKR gene. As a result, typical systemic viral symptoms were observed in all plants within 14 days (see FIG. 4 ). In contrast, 5 out of 16 cases of transgenic plant strains showed late symptoms, and series # 14 showed the highest viral resistance (Table 1).

TEV caused systemic infection symptoms showing vein necrotic symptom in control plants ( FIG. 4 ). In addition, all of the five transgenic plant lines that showed resistance to CMV showed obvious resistance to TEV (Table 1), and the resistance of series # 14 was as high as that for CMV.

Resistance of Transgenic Plants to Viral Infections line# Percentage of plants inoculated with viral symptoms CMV TEV * dpi 7 11 15 19 8 12 17 25 Control 78 100 100 100 17 100 100 100 3 32 32 49 67 12 35 49 62 8 49 92 92 92 26 57 57 57 10 35 46 58 72 8 78 78 78 12 19 70 70 70 20 55 59 59 14 0 0 40 40 15 38 38 38

* dpi (days after post-inoculation)

In the above CMV is a result of inoculation by diluting the plant extracts infected with CMV by 15 times, TEV is a result of inoculation by diluting 30 times the plant extracts infected with TEV.

Surprisingly, an average of 60% and 62% of the five transgenic plant lines showed no viral infection symptoms two months after inoculation with CMV and TEV, respectively. These series also showed resistance to PVY.

Example 4 ELISA Analysis

ELISA analysis was performed to investigate the accumulation of virus particles in the transgenic plants inoculated with CMV.

Leaf tissues were ground under phosphate buffer solution (PBS) containing 0.05% Tween 20 (PBST), virus particles were extracted and diluted to use for ELISA analysis. Plates were coated with 50 μl of each dilution and then incubated overnight at 4 ° C. The plate was then washed three times with PBST.

The reaction was terminated by treatment with blocking solution (PBST with 3% BSA) and then reacted with a polyclonal antibody specific to CMV (DSMZ # AS-0196, Germany). After the plate was washed, anti- IgG bound to alkaline phosphatase was added as a secondary antibody and reacted at room temperature for 2-3 hours. After washing to remove the unbound antibody, para-nitrophenyl phosphate (PNPP) disodium solution (1 mg / ㎖) was added and incubated at room temperature under dark conditions. Then, the absorbance was measured at 405 nm to investigate the degree of reaction.

As a result, after 24 hours of inoculation of the virus, the concentrations of the antigens in the control and the leaves of the transgenic plants were found to be similar to both (see FIG. 5 ). However, after 48 hours, the extent of virus accumulation in transformed series # 14 was lower than that of the control group. On the other hand, 15 days after infection, the virus titer was measured on the upper leaves, and the antigen concentration of the transgenic plants that did not exhibit the symptoms of viral infection after inoculation with CMV was similar to that of the negative control plants inoculated with only the medium (see FIG. 6 ). . These results indicate that a lot of viruses do not accumulate in the transgenic plants that do not show symptoms of viral infection.

In the leaves of the transformants exhibiting symptoms of viral infection, virus accumulation was lower than that of the control group. Thus, it can be seen that the PKR gene in transgenic plants effectively controls viral replication and / or proliferation, thereby imparting not only simple viral resistance but also substantial viral resistance to the plant.

Example 5 Phosphorylation Assay

In vitro autophosphorylation assays were performed using leaf tissue extracts from control and transgenic plants to investigate whether the introduced human PKR gene functions properly in tobacco and can change phosphorylation status at the protein level. It was.

First, 24 hours after grinding, whole protein was extracted from leaves of Samsun NN and PKR transgenic plants. Leaf tissues were mixed solution of 100 mM tris-hydrochloric acid (pH 7.5), 10% glycerol, 5 mM dithiothreitol, 100 mM potassium chloride, 1 μg / ml apotinine, 1 μg / ml leupeptin, 2 mM phenylmethylsulfonyl fluoride Homogenized at After 10 μl of the total protein solution was left at 30 ° C. for 10 minutes, phosphorylation buffer solution containing 5 μM of [γ- ³² P] ATP (100 ci / mmol) and poly r (I) -r (C) at different concentrations. (20 mM Tris-HCl, pH 7.4, 5 mM MgCl ₂ , 5 mM MnSO ₄ ) was added to a final volume of 50 μl. After the reaction was terminated by adding twice the concentrated electrophoretic sample buffer, each sample was separated by 10% SDS-PAGE, and confirmed by magnetic radiograph.

When extracts from control plants were incubated, it was found that ³² P was introduced at basal level from Mr [γ- ³² P] ATP to Mr 68,000 (p68). In addition, the phosphorylation level of p68 in control plants was increased by the addition of ds RNA. A similar pattern showing an increase in p68 phosphorylation by ds RNA was also observed in extracts of plants transformed with PKR . However, as shown in the autodiagram using a densitometer, the inflow of ³² P into P68 from the PKR transformant was found to be more than two times higher than that of the control group (see FIG. 7 ). .

In addition, the shift to the upper band by phosphorylation was relatively higher in PKR transformants than in the control. The upper band here represents the phosphorylated protein and the lower band appears to represent either unphosphorylated form or degraded product.

Combining these results, it can be seen that the introduced PKR gene is normally expressed in the transgenic plant, and the expression of these genes has an effect of increasing the degree of phosphorylation of p68 in the PKR transgenic plant.

On the other hand, it was confirmed that the translation of bromide mosaic virus in wheat embryo hydrolyzate was inhibited by the addition of activated PKR in vitro (Langland et al ., The Journal of Biology Chemstry 271 : 4539-4544, 1996). Therefore, since the introduced PKR gene exerts its function in the transgenic plant, it is judged that the PKR transgenic plant can suppress the proliferation of the virus.

Plants transformed with the PKR gene of the present invention exhibit complex resistance to most plant viruses with ds RNA as replication intermediates, and inhibit growth, loss of fertility and Side effects such as abnormalities do not appear. In addition, viral resistance is expressed throughout the whole body as well as the site of viral infection, there is no risk of emergence of recombinant viruses that can occur when using a virus-derived gene can be useful as a safe way to give a complex virus resistance to useful plants Can be.

Claims (9)

A method of conferring a complex virus resistance to a plant by transforming the plant with a recombinant vector for plant transformation comprising a gene encoding a dsRNA-dependent protein kinase (PKR) activated by double-stranded RNA. The method of claim 1, wherein the PKR gene is derived from an animal or yeast. The method of claim 2, wherein the PKR gene is derived from a human. The recombinant vector of claim 1, wherein the recombinant vector comprises an Arabidopsis blue copper binding protein promoter (BCB promoter) that rapidly activates transcription by a wound or viral infection, and a PKR gene associated with the promoter. A method of imparting complex virus resistance to plants. The method of claim 4, wherein the recombinant vector is pBCB-PKR (Accession No .: KCTC 0823BP). The method of claim 1, wherein the plant to be transformed is selected from the group consisting of food crops, vegetable crops, special crops, fruit trees, flowers and feed crops. The food crop according to claim 6, wherein the food crops are rice, wheat, barley, corn, soybeans, potatoes, wheat, red beans, oats, sorghum; Vegetable crops include arabidopsis, Chinese cabbage, radish, red pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin, green onion, onion, carrot; Special crops include ginseng, tobacco, cotton, sesame, sugar cane, sugar beet, perilla, peanut, rapeseed; Fruit trees include apple trees, pears, jujube trees, peaches, pears, grapes, citrus fruits, persimmons, plums, apricots, bananas; Flowers include roses, gladiolus, gerberas, carnations, chrysanthemums, lilies, tulips; Forage crops are selected from the group consisting of lygras, red clover, orchardgrass, alpha alpha, tol pesqueu, perennial lyse, and the like. The method of claim 1, wherein the virus resistance is characterized in that the plant is characterized in that it is resistant to at least one virus selected from the group comprising a cucumovirus group (CMV), a potyvirus group (TEV), a PVY (potyvirus group), complex virus resistance to plants How to give. Recombinant vector pBCB-PKR for plant expression introduced into plants to confer complex virus resistance (Accession No .: KCTC 0823BP)