KR100314837B1 - Coat gene of carnation mottle virus(carmv) and recombinant binary vector containing the same - Google Patents

Abstract

PURPOSE: A coat gene of Carnation Mottle virus(CarMV) and a recombinant binary vector containing the same are provided, thereby producing carnation or other crops having tolerance to Carnation Mottle virus(CarMV). CONSTITUTION: A coat gene of Carnation Mottle virus(CarMV) has the nucleotide sequence shown in figure 2. A recombinant vector pOST 8002 is constructed by inserting the coat gene of Carnation Mottle virus(CarMV) into a vector pTRL. A recombinant vector pOST 8003 for transforming a plant with the recombinant vector pOST 8002 is constructed by inserting the coat gene of Carnation Mottle virus(CarMV) into a binary vector pGA482 for transforming a plant.

Description

A coat protein gene of carnation mottle virus and a recombinant binary vector containing the same.

The present invention relates to envelope genes of Carnation Mottle Virus (hereinafter, simply referred to as 'CarMV') and biologic vectors for transformation necessary for the development of crops containing the same and resistant to CarMV.

Currently, the production of domestic flowers is about 400 billion Won, of which the production of cut flowers is about 150 billion Won (Ministry of Agriculture, Forestry and Fisheries, 1993). One of the biggest factors in the cultivation and production of these crops is the viral disease, which is so serious that annual damages amount to several hundred billion won in Korea. Viral disease is difficult to classically resistant breeding breeding, and unlike other diseases, it is also difficult to control by drugs. Therefore, there are methods to control viral pathogens in order to use disease-free grafts or to reduce disease. In recent years, the development of antiviral crops based on plant transformation technology, which is a developmental product of plant genetic engineering, has been studied extensively and the results have been evaluated positively.

Genes useful for transformation of plants required for the production of antiviral crops include virus replication genes, coat protein genes, satellite RNA genes, reverse transcription genes, ribozymes, and ribosome inactivation protein genes. Therefore, the isolation and effective use of these useful genes will control the viral diseases of crops and prevent the growth of farm income as well as maintaining stable national economic base.

Among the various flowers, carnation is one of the three major crops in Korea, along with chrysanthemums and roses, and is suffering from viruses and infections. As of 1992, carnation has a total area of 8lha, about 5% of the total area, about 8% of production, and about 10% of production ('92 flower cultivation status, Ministry of Agriculture, Forestry and Fisheries, 1993). Currently, carnation viruses in Korea are carnation mottle virus, carnation italian ringspot virus, carnation latent virus, carnation ring spot virus and carnation vane mottle virus. Among them, carnation mottle virus is the most widely infected, It has been reported to cause many harmful symptoms.

In order to overcome the viral diseases of carnation, breeding has been attempted to introduce virus resistance factors from wild type, but the development of carnation varieties which are resistant to viruses while retaining the existing quality has not been developed yet. In order to overcome the above-mentioned problems, methods of using plant genetic engineering for the development of crops using transgenic technology are emerging.

In Korea, in 1993, the RDA applied for a patent under the name of 'Lybozyme Imhexane fragment for the development of my viral crops, its manufacturing method and usage', and also applied 'Cucumber mosaic virus coat protein gene' (Applicant Upjohm company / Domestic Publication No. 90-700604 / published June 29, 1989).

In order to prepare antiviral carnation cultivars, it is possible to use various genes mentioned above. In the present invention, however, a coat gene has been specifically selected. These techniques include the introduction of the envelope gene of tobacco mosaic virus into tobacco, (Bevan, MW et al., EMBO J. 4, pp 1921-1926 (1985), Powsll, AP, et al., Science 232, pp 738-743 (1986) )). Alfalfa Mosaic Virus (Tumer et al., EMBO J. 6, pp 1181-1189 (1987), Loesch-Fries et al., In "Molecular Strategies for Crop Protection", pp 221-234 (1987), van Dun et al., Virology 163, PP 572-578 (1987), Tobacco rattle virus (Van Dun et al., Virology 159, pp 299-305 (1987)), cucumber mosaic virus , potato virus X (Hemenway et al., EMBO J. 7, pp 1273-1228 (1988)), tobacco streak virus (Tobacco streak virus) virus: van Dun et al., Virology 164, pp 388-389 (l988)) have been reported to produce antiviral crops.

In view of the various reports reported so far, the envelope genes of various viruses are effectively resistant to viruses. Therefore, the inventors of the present invention have found that the tobacco mosaic virus t strain (TMV-t), capsular mild mottle virus (PMMV) And then cloned the envelope gene of CarMV, which is known to cause serious damage to the carnation.

Accordingly, it is an object of the present invention to provide a coat gene of CarMV which can be used to develop carnation or other crops resistant to Korean CarMV.

Another object of the present invention is to provide a recombinant binary vector comprising the gene and usable for transformation of plant cells.

According to an aspect of the present invention, there is provided a carnation mottle virus (CarMV) coat protein gene having the nucleotide sequence shown in FIG.

According to another aspect of the present invention, there is provided a recombinant vector pOST 8002 in which the envelope gene is inserted into a vector containing an expression cassette capable of expressing in a plant.

According to another aspect of the present invention, there is provided a recombinant Binary vector pOS7 8003 comprising the envelope gene in a vector for expression and transformation of plant body.

According to the Budapest Treaty, the following microorganisms have been deposited with the Korean Society of Bacterial Kidnap, Incorporated, located at 134, Shinchon-dong, Seodaemun-gu, Seoul, and their accession numbers have been assigned to KFCC-I0897 and KFCC-I0898, respectively.

Hereinafter, the present invention will be described in detail.

The research team secured a large amount of the carnations showing the carnation mottle virus symptoms through the pavement survey in the carnation cultivation complex in the Gyeongju province, and purified the Korean CarMV from it. Genomic RNA was isolated from the purified Korean type CarMV and complementary DNA (cDNA) was synthesized using a random hexamer of a TimcSaver cDNA synthesis kit (purchased from Pharmacia) as a primer. Were synthesized. The nucleotide sequence of the synthesized cDNA was partially determined and compared with the nucleotide sequence information of the data bank. As a result, it was confirmed that the Korean type CarMV was the same virus as the existing CarMV.

CarMV is divided into the group of Carmo virus group because its whole genome is composed of replicase, movement protein and coat protein. An oligonucleotide (CarMV) prepared by referring to the nucleotide sequence at the 3'end of the base sequence of CarMV (Morris et al., Nucleic Acids Res. Vol. 13 No. 18: 6663-6677 (1985) 3 ': 5'-GCCGTGTGTGTCTAGACAAA-3'OH: 20 nucleotides) using CarMV genomic RNA (RNA) as a template and reverse transcriptase to a single stranded complementary deoxy-ribohexane (DNA) was synthesized by adding E. coli RNase H and E. coli I DNA polymerase I to a complementary deoxyribonucleic acid (hereinafter referred to as "cDNA"). With reference to the reported nucleotide sequence, oligonucleotides complementary to the 3 'and 5' side were cloned to clone the 2657th base to the 3748th base containing the coat gene of CarMV (the cistron of the CarMV envelope gene was 2669-3715) 3 'side: 20 nucleotides / 5' side: 20 nucleotides) were synthesized. A known polymerase chain reaction was carried out using the synthesized oligonucleotide and the previously synthesized double-stranded CarMV cDNA mixture template. The resultant synthetic DNA (DNA) product was cut out using restriction enzyme recognition sites of Nco I and Xba I formed by oligonucleotides used in the chain polymerization, and DNA fragments Was introduced into a vector pTRLII containing a CaMV-35S promoter-TEV 5 'leader-CaMV 35S terminator cassette expressible in the body. Subsequently, only the gene expression cassette site was cut and introduced into the Agrobacterium-mediated binaly vector for transformation into the plant in the above recombinant vector.

This binary vector was transformed into Agrobacterium, and the nucleotide sequence of the cloned CarMV envelope gene was determined.

Hereinafter, the present invention will be described more specifically based on examples.

Example 1 Genomic RNA (RNA) Isolation from Purely Isolated Korean CarMV

The virus of CarMV dissolved in 0.01 M sodium phosphate buffer (300 μl) was mixed with an equal volume of (300 μl) phenol: chloroform: isoamyl alcohol (24: 24: 1: v) For 5 minutes and the supernatant was transferred to a new test tube three times. To the final supernatant, 1/10 volume of (30 μl) sodium acetate (pH 6.0) and 300 μl of iso-propanol were added and kept at minus 70 ° C. (-70 ° C.) for 20 minutes. This was centrifuged at 12,000 g for 10 minutes, and the supernatant was discarded. Only the precipitate was washed with 0.5 ml of 70% ethanol, vacuum-dried, and dissolved in 30 μl of distilled water.

Example 2: Synthesis of sidiane (sDNA) from genomic ELNA (RNA) of Korean CarMV

(CDNA) was synthesized from the CarMV genome RNA (RNA) obtained in Example 1 using a time-saving CDNA synthesis kit. 5 μg of CarMV genome RNA (RNA) and distilled water not contaminated with RNase were added to the test tube, and the volume was adjusted to 20 μl volume. After denaturation at 65 ° C for 10 minutes, the reaction mixture was immediately transferred to ice, Lt; / RTI > The above reaction mixture was mixed with first strand reaction mixture (first strand reaction mixture) in which the reverse transcriptase, BSA, dATP, dCTP, dGTP and dTTP were dissolved in an aqueous solution, and 1 μl of DTT (200 mM) And 1 μl (0.5 mg / ml) of a random hexamer of a time-saving CDNA synthesis kit as a primer were added and reacted at 37 ° C. for 1 hour. The entire reaction mixture was transferred to a second strand reaction mix (RNasc H, E. coli DNA polymerase I and dNTPs), and then transferred to a second strand reaction mixture ℃ for 1 hour. After the above reaction was completed by heating at 65 ° C for 10 minutes, 100 μl of phenol: chloroform (1: 1 / v) was added and centrifuged for 1 minute. Then, 100 μl of the aqueous solution And purified using a spun column (included in the kit). To clone the purified DNA (cDNA) into a pBluescript SK (+) vector, 1 μl of an Eco RI / Not adapter (0.5 μg) in which a restriction enzyme recognition site was introduced and a polyethylene glycol buffer solution , 1 μl of 20 mM ATD, 1 μl of ligase (10 enzyme units), reacted at 16 ° C. for 1 hour, and then heated at 65 ° C. for 10 minutes to quench the reaction. 1 μl of 70mN ATP and 1 μl of T4 polynucleotide kinase (10 units) were mixed and reacted at 37 ° C for 30 minutes. The reaction product was heated at 65 ° C for 10 minutes to denature and terminate the reaction enzyme, followed by centrifugation for 10 minutes by adding 140 μl of phenol: chloroform (1: 1 volume) (CDNA) with an Eco RI / Not I adapter attached to a blue script plasmid (PBluescript) SK (+) vector digested with Eco RI restriction enzyme 15 ㎕ of a spun column eluate of the above reaction, 10 연결 of a coupling reaction buffer (66 mM Trsi-HCl (pH 7.6), 0.1 mM spermidine, 6.6 mM MgCl ₂ , 10 mM DTT and 150 mM NaCl) , 10 μl of 3 mM ATP and 1 μl (10 units) of T4 DNA (DNA) ligase were mixed together, followed by addition of 100 μl of a blue Script plasmid pBluescript SK (+) vector digested with Eco RI restriction enzyme (Final volume: 35 占 퐇) and reacted at 16 占 폚 for 1 hour. The reaction product DNA (DNA) was introduced into Escherichia coli (DH5a) by electrophoretic transformation (see Maniatis et al., Molecular cloning, A laboratory manual, 2nd ed. 1.75, CSH press, 1989). The cells were plated on LB solid medium (10 g of bacto-tryptone per liter, 5 g of enzyme extract, 10 g of sodium chloride and 15 g of bacto-agar) containing 50 mg / ml of ampicillin and reacted at 37 ° C for 12 to 16 hours to contain a recombinant vector Ten colonies were selected.

Plasmids were isolated and purified from the above 10 colonies by an alkaline lysis method (Maniatis et al., "Molecular Cloning" 2nd ed. 1.21, CSH press, 1989). The plasmid thus isolated and purified was digested with Eco RI restriction enzyme, and subjected to agarose gel electrophoresis to confirm that a cDNA fragment synthesized from CarMV RNA (RNA) was inserted into the vector. The size of the cDNA fragment varied from 500 bp to 2 kbp. A commercial primer of the pBluescript SK (+) vector was prepared by the method of determining the base sequence by the dideoxy method of Sanger (Sanger, et al., Proc. Natl, Acal, Sci. USA 74: 5463 Were used to determine the cDNA nucleotide sequences synthesized. As a result of determining approximately 150 base sequences from both ends of the cDNA, it was confirmed to be similar to the base sequence of CarMV (E. Alonso et al., J. Gen. Virol. 72, pp. 2875-2884, 1991) .

Soon, the following experiment was performed to clone only the coat gene of Korean carMV.

Example 3: Amplifying the envelope gene from genomic RNA (RNA) of Korean CarMV.

In order to clone the necessary envelope operator in the genomic RNA (RNA) of CarMV obtained in Example 1, the nucleotide sequences of the 5 'and 3' side of the envelope gene were referred to from the genetic information obtained in Example 2, Oligonucleotides were synthesized.

0.5 μg (8 ml volume) of Genomic RNA (RNA) of CarMV was denatured by heating at 65 ° C for 10 minutes, and then transferred to ice to fix the denatured state. CarMV Genomes To a test tube prepared for synthesis of the first strand of cDNA from RNA (RNA), 4-5 μg of CarMV genomic RNA (RNA) and 1 μl of murine reverse transcriptase (10 enzymes 1 μl of 20 mM dNTP, 1 μl of 0.1 M DTT and 0.5 μg of CarMV 3 'oligonucleotide were mixed and reacted at 37 ° C. for 1 hour. After the reaction, the RNA-cDNA duplex was heated at 90 DEG C for 5 minutes, and strands were separated. Then, the double-stranded cDNA synthesized for amplification of the coat protein gene together with the synthesis of the second strand cDNA was denatured and fixed, , 5 μl of a 10-fold polymerase chain reaction (PCT) buffer solution, 1 μl of 20 mM dNTP mix, 1 μl of CarMV 3 'oligonucleotide, 200 μl of CarMV 5' oligonucleotide, 200 mg of Vent DNA polymerase (2.5 enzyme units) in a 0.5 ml PCR tube and allowed to reach a final volume of 50 μl with distilled water. On top of that, mineral oil (50 μl) was gently added without mixing with the solution in the lower layer, and then reacted in a thermal cycler (MJ research, Model TM 100) under the following conditions.

The envelope gene of CarMV amplified by the described reaction was confirmed by electrophoresis on 0.7% agarose gel and the results are shown in FIG. In the first lane of the figure, the DNA of the lambda phage (300 mg) was digested with restriction enzyme Bst EII and electrophoresed to compare the size of the amplified CarMV cDNA. The second lane was precisely amplified at a size of about 1.1 kilo basepair, which was supposed to amplify the envelope gene by the polymerase chain reaction of CarMV cDNA purchased from American Type Culture Collection (ATCC). And we could amplify the envelope gene fragments of the same size from CarMV isolated from Korea. The reaction mixture was mixed with the same volume of chloroform, centrifuged at 12,000 xg for 5 minutes, transferred to a new test tube twice, and then washed with 1/10 volume of sodium acetate sodium acetate, pH 6.0) and 2 volumes of ethanol. Centrifuged at 12,000 x g, vacuum dried, and dissolved in 30 μl of TE Buffer.

Example 4: Preparation of vector pOST 8002 and transformation of Escherichia coli

The restriction enzyme recognition site in the envelope gene of CarMV obtained in Example 1 (restriction enzyme recognition site linkable to the prepared oligonucleotide: CarMV 3 '; Xba I / CarMV 5' Nco I site) Nco I and Xba I. 300 mg of a coat gene obtained by digesting 1 μg of a vector pTRL II having a Dual CaMV-35S promoter-TEV reader-35S terminator with Nco I and Xba I was mixed, and the T4 DNA (DNA) ligase was ligated as a first enzyme unit, 2 [mu] l of buffer solution, 2 [mu] l of 10 mM ATP, and then adjusted to a final volume of 20 [mu] l with distilled water. The mixture was reacted at 16 ° C for 15 hours to allow the coat gene to be inserted between the Nco I and Xba I sites of the pTRL II vector. Transformation of the DNA into E. coli (DH5a) was carried out by the calcium chloride method (Maniatis et al., Molecular Cloning, A laboratory manual, 2nd ed. 1.82-1.84, CHS , 1989) were plated on LB solid medium containing 50 mu g / ml of ampicillin and cultured at 37 DEG C for 12 to 16 hours to select colonies containing the recombinant vector.

Plasmids were isolated from the selected colonies by the above-described alkali dissolution method and purified pure by the CaCl 2 concentration gradient ultracentrifugation method (see the same document, 1.42 -1.45). The separated and purified polystyme was digested with restriction enzymes such as Nco I and Xba I and then subjected to agarose gel electrophoresis to confirm that the CarMV envelope gene fragment was inserted into the vector, and this was named [pOST 8002] 5).

Example 5: BlueScript plasmid (pBluescript) Determination of nucleotide sequence of CarMV envelope gene introduced into SK (+) vector

The nucleotide sequence of the gene synthesized by the nucleotide sequencing method of Sanger's dideoxy method was confirmed using commercial primer (T7, T3 primer set) of Blue Script plasmid (pBluscript) SK (+), Respectively.

Example 6: Preparation of vector pOST 8003 and transformation into Agrobacterium LBA 4404

Experiments were carried out to produce pOST 8002 prepared in Example 4 in a form that can be introduced into a plant. The expression cassette (part of the CaMV Dual 35S promoter-TEV leader-coat gene-35S terminator) in the pOS7 8002 plasmid was digested with HindIII restriction enzyme to re-insert into pGA482. 1 μg of vector PGA482 was digested with Hind III and then digested with Hind III to obtain 200 μg of the expression cassette fragment isolated from the pOST 8002 plasmid. The resultant plasmid was mixed with 2 μl of T4 DNA (ligase) , 2 μl of 10 mM ATP was added, and the final volume was adjusted to 20 μl with distilled water. The mixed product was reacted at 16 ° C for 15 hours to insert a coat gene between the Hind III recognition sites of the pGA482 vector. Transformation of the above-mentioned DNA (E. coli) into E. coli (DH5?) Was carried out by the calcium chloride method, and was plated on LB solid medium containing 13 占 퐂 / ml of tetracycline and reacted at 37 占 폚 for 12 hours for 16 hours to obtain a recombinant vector Colonies were selected.

Blasmid was isolated from the selected colloid by the above-mentioned alkali dissolution method, and further purified by CsCl concentration gradient ultracentrifugation method. After the cleavage of the fibrid with the Hind III restriction enzyme, the carMV envelope fragment was inserted into the pGA482 vector by agarose gel electrophoresis. The plasmid into which the CarMV envelope gene was introduced was named [pOST 8003], which is shown in FIG.

Transformation of the expression vector into Agrobacterium LBA4404 was carried out by the freeze-thaw method (Stanton B. Gelvin et al., Plant Molcular Biology manual, 1988, pp: PMAN-A3 / 7 ) Colonies containing a vector prepared by plating on YEP solid medium (10 g of bacto-peptone per liter, 10 g of enzyme extract, 5 g of sodium chloride, 15 g of bacto-agar) containing tetracycline at 28 캜 for 48 hours at 28 캜 Respectively.

Plasmids were isolated from the colonies by the Agrobacterium plasmid quick-screen method (Stanton B. Gelvin et al., Plant Molecular Biology manual, 1988, pp: PMAN-A3 / 9). The plasmid thus isolated and purified was digested with HindIII restriction enzyme and then subjected to agarose gel electrophoresis to confirm that the CarMV envelope gene fragment was inserted into the pGA482 vector.

FIG. 1 shows the nucleotide sequences of two primers synthesized for cloning only the coat gene of carnation mottle virus in a whole nucleotide sequence by a sequential polymerase chain reaction method, and a coat gene amplified therefrom

FIG. 2 is a diagram showing the nucleotide sequence of the carnation mottle virus envelope gene according to the present invention

Figure 3 shows the sequence of the amino acid sequence encoded by the second polynucleotide sequence

FIG. 4 is a schematic diagram showing the structure of vector pOST 8001 in which the complementary DNA (c DNA) of the carnation mottle virus envelope gene is inserted into a Bluescript plasmid vector (pBluescript) Sk (+) to determine a base sequence

5 shows the expression of the carnation mottle virus envelope gene in the plant by the Nco I restriction of the vector pTRL II under the control of the cauliflower mosaic virus (CaMV) -35S promoter, the TEV 5 'leader sequence and the cauliflower mosaic virus 35S terminator A schematic diagram showing the structure of vector pOST 8002 in which the envelope gene of carnation mottle virus is linked between the enzyme and the Xba I restriction enzyme recognition site

FIG. 6 is a schematic diagram showing the structure of vector pOS7 8003, which is ligated between Hind III recognition sites of vector pOST 8002 to introduce the gene of the present invention into Agrobacterium LBA 4404

Claims (3)

A carnation mottle virus envelope gene having the nucleotide sequence shown in FIG. 2 A recombinant vector pOST8002 in which the carnation mottle virus envelope gene of claim 1 is inserted into a replicable vector capable of expressing. A recombinant vector POST 8003 in which the carnation mottle virus envelope gene of claim 1 is inserted into a plant transformation viral vector for expression in a plant body.