KR100250901B1 - Nucleotide sequence of Cymbibium Mozaic Virus, Expressing Vector and Transgenic Plant Thereof - Google Patents

Abstract

PURPOSE: Provided is an expression vector to transform Agrobacterium with genes of Cymbidium mosaic virus (CyMv) and to develop transformed plants resistant against CyMv using the bacterium. CONSTITUTION: An expression vector for transforming Agrobacterium with genes of Cymbidium mosaic virus (CyMv) is characterized by the next steps of: i) cutting pCyMV1 with restriction enzymes Pstl and EcoR1 and treating segments with T4 DNA polymerase to obtain blunt segments; ii) eluting bands containing genes of envelop proteins through electrophoresis; iii) ligating the bands with vector Smal-cut pGEM-7Z(+) and transforming E. coli JM109 with the vector; iv) cutting DNA obtained from the bacteria with BamH1 and PuvII to check the insertion direction of envelop proteins (pCyCPS is in sense direction and pCyCPA in antisense direction); v) cutting pCyCPS with Xbal and eluting bands containing genes of CyMV envelop protein; vi) ligating segments with Xbal-cut pMBPl and transforming E. coli JM109 with the vector; vii) checking the direction of insertion proteins (Clones obtained here are pMBPCPS and pMBPCPA); viii) cutting pMBPCPS and pMBPCPA with HindIII and XbaI (pMBPCPS/J is in sense direction and pMBPCPA/J in antisense direction).

Description

Nucleotide sequence of Cymbibium Mozaic Virus, Expressing Vector and Transgenic Plant Thereof}

The present invention relates to a nucleotide sequence, an expression vector, and a transformant of an orchid plant (Cymbidium) mosaic virus (CyMV).

Among the monocotyledonous plants, the variety and cultivation area of orchid plants are steadily increasing due to the flower's splendor and subtle scent. However, orchid is a plant that mainly targets flowers and stems, and once it is damaged by insects, its commodity value decreases and must be discarded. Of the pest damage, the most important deterioration of orchid quality is mosaic virus disease, so far about 25 kinds of orchid viruses are known. Among them, Cymbidium mosaic virus (CyMV) and Odontoglossum ring spot virus (ORSV) have been mainly studied. Especially, the disease caused by CyMV is widely distributed around the world and the damage is the most severe.

Therefore, various studies have been conducted to reduce the virus infection and its damage to orchids, but the effect is still insignificant. The reason is that the virus infection mechanism is uncertain. However, in order to produce a virus-resistant plant by genetic engineering techniques, a method of inserting the envelope protein gene into the plant, using satellite RNA, a subgenomic viral nucleic acid, and finally inserting antisense RNA to inhibit Viral gene expression You can consider the method.

Therefore, the present invention decided to introduce the first of the various methods, determine the size of the CyMV genome and envelope proteins, synthesize cDNA from CyMV RNA, and then prepare subclones to determine the base sequence of CyMV 3 '. For that purpose. It is another object of the present invention to provide a vector for developing CyMV resistant transformant plants by separating the envelope protein gene, inserting it into a plant expression vector, and transforming it into Agrobacterium.

Hereinafter will be described in detail so that those skilled in the art can easily implement the specific configuration and operation of the present invention.

1 is a schematic diagram showing the structure of pCyMV1.

CyMV cDNA has been shown to be synthesized from CyMV RNA and linked with EcoRI-Cut pBluescript KS vector.

Figure 2 is a schematic diagram for determining the restriction map and nucleotide sequence of pCyMV1.

Among the restriction enzymes are E: EcoRI, P: PstI, and Pv: PvuII M: MspI.

Figure 3 is a base sequence of cDNA containing the envelope protein gene of CyMV.

Square marks indicate start codon and end codon.

Figure 4 is a schematic diagram showing the plant expression vector pCyCPS and pCyCPA production process obtained by cloning the CyMV envelope protein.

The restriction enzyme symbol is as follows.

B: BamHI, E: EcoRI, K: KpnI, P: PstI, Pv: PvuII, S: SmaI, X: XbaI

Figure 5 is a schematic diagram of the production of plant expression vector beta pMBPCPS and pMBPCPA in which CyMV envelope protein is inserted

A: Structure of pCyCPS digested with restriction enzyme XbaI

B: Structure of the plasmid vector pMBPI

C: The above two vectors for plant expression

Figure 6 is a photograph showing the results of agarose gel electrophoresis of the plant expression vector pMBPCPS / J and pMBPCPA / J.

Lane M: Lamda HindⅢ size marker

Lane I: pMBP I cleaved with KpnI + HindIII

Lane 2-3: pCyCPA digested with KpnI + HindIII

Lane 4-5: KpnI

Figure 7a is a photograph showing the results of agarose gel electrophoresis of plant expression vectors pMBPCPS / L and pMBPCPA / L.

Lane M; Lamda HindⅢ size marker

Lane 1: pCyMVI digested with EcoRI

Lane 2: pMBPI digested with KpnI + HindⅢ

Lane 3-4: pMBPCPS / L digested with KpnI + HindⅢ

Lane 5-6: pMBPCPA / L digested with KpnI + HindⅢ

Figure 7b is a photograph showing the results of performing Southern blot using the envelope protein gene of CyMV as a probe.

Lane M; Lamda HindⅢ size marker

Lane 1: pMBPI digested with KpnI + HindⅢ

Lane 2-3: pMBPCPS / J digested with KpnI + HindⅢ

Lane 4-5: pMBPCPA / J digested with KpnI + HindⅢ

Lane 6: pCyCPS digested with KpnI + HindⅢ

Bacterial strain

The strain used in the experiment was E. coli JM109 (recAl supE44 endAl hsdR17 gyrA96 relAl thiΔ lac-proAB) F '[traD36 proAB ⁺ lacl ^a lacZΔM15]. For the production of plant expression vectors, one of the Agrobacterium strains was used. LBA4404 was used.

Plamid

pBluscript KS (Stratagene) and pGEM-7Z (+) (Promega) were used for cDNA cloning and subcloning, and pMBP I was used as a plant expression vector.

virus

CyMV particles used in this experiment were distributed in the Department of Agricultural Biology and Virology, Seoul National University.

Culture medium

LB medium (1% tryptone, 0.5% yeast extract, 1% NaCl) was used to culture E. coli JM109, and ampicillin (50 μg / ml), X-gal (20 mg / ml), LB medium containing IPTG (200 mg / ml) was used. pMBP I was cultured in LB medium containing kanamycin (50 μg / ml), Agrobacterium was cultured in YEP medium (1% peptone, 1% yeast extract, 0.5% NaCl), and kanamycin (50 μg / ml) was used to select transformants. YEP medium containing ml) was used.

Reagents, Enzymes Used

cDNA synthesis system, Klenow polymerase, T4 DNA polymerase, T4 DNA ligase, T4 DNA kinase were purchased from Promega and BamHI, EcoRV, HindIII, Msp I, PstI, PvuII, SacI, SmaI, XbaI, KpnI were purchased from BM. .

ECL direct nucleic acid labeling and detection system kits and nylon membranes were purchased from Amersham, sequencing kits from USB, tryptone, yeast extract, and agar from DIFCO. Other reagents, including agarose, were purchased from SIGMA Chemical Co. and Fluca.

Hereinafter, the size of the CyMV envelope protein will be determined and the CyMV cDNA synthesis process will be described with examples by process.

Step 1: CyMV Envelope Protein and Gene Size Analysis

Size Analysis of CyMV Envelope Proteins

The size of CyMV envelope protein was analyzed by electrophoresis of 12 μg of CyMV particles on 10% sodium dodecyl sulfate-polyacrylamide gel and stained with Coomassie Brilliant Blue. As a result of SDS-PAGE, the envelope protein of CyMV used in this experiment was about 26 kDa.

Purification and Sizing of CyMV RNA

5 µl of extraction buffer (4M guanidium thiocyanste, 50 mM Tris pH 7.0, 10 mM EDTA, 5 mM sodium citrate 0.1 M β-Mercaptoethanol) 400µg 10% sarcosyl was added to 100µg of CyMV particles and heated at 65 ° C for 5 minutes. The same volume of phenol / chloroform / isoamyl alcohol was added thereto, shaken, soaked in ice for 10 minutes, and centrifuged at 12,000xg for 10 minutes at 4 ° C. Take an aqueous layer, add the same volume of chloroform / isoamyl alcohol, shake and centrifuge for 10 minutes at 12,000xg at 4 ℃. Take an aqueous layer, add 0.1 times volume of 3M sodium acetate (pH5.2), add 2.5 times volume of ethanol, mix well, store for 1 hour at -20 ℃, centrifuge at 12,000xg for 20 minutes, and discard supernatant. Washed once with ethanol and lyophilized. This pellet was dissolved in distilled water treated with DEPC. Purified RNA was placed on 1% formaldehyde agarose gel to measure CyMV RNA size and OD ₃₆₀ to determine the amount of CyMV RNA. The size of CyMV genome was about 6.2kb.

Second Step: CyMV cDNA Synthesis and Selection

CyMV cDNA Synthesis

CyMV synthesis was followed by Gubler and Hoffman (1983). First strand synthesis was performed by mixing 2 μg of mRNA with 1 μg of XbaI primer adaptor, adjusting the volume to 15 μl, heating at 70 ° C. for 5 minutes, cooling to room temperature, and then reacting with a total 25 μl reaction solution [50 mM Tris-HCI (pH8). .3), 50 mM KCl, 10 mM NaCl2, 0.5 mM spermidine, 10 mM DTT, 4 mM sodium pyrophosphate, 25 units of rRNasin, 1 mM dATP, dCTP, dGTP, dTTP, 30 units of reverse transcriptase] The reaction was carried out for 60 minutes.

Second strand cDNA synthesis was performed using 100 μl of reaction solution (50 mM Tris-HCl / pH 7.6, 100 mM KCl, 5 mM MgCl ₂ , 50 μg / μl bovine serum albumin, 5 mM DTT, 8) ㎍ / ㎖ RNase H, 230㎍ / ㎖ E. coli DNA polymerase I) in the row was in the 14 ℃ for 4 hours, first strand, respectively 5㎍, taken separately ssikeul 10㎍ [α- ³² of second strand synthesis from 5-10μCi After inserting P] dCTP and performing a tracer reaction, the degree of synthesis was confirmed by autoradiogram. The synthesized cDNA was treated at 70 ° C. for 10 minutes, 2 units of T4 DNA polymerase was added and reacted at 37 ° C. for 20 minutes to form a blunt end. 10 μl of 200 mM EDTA was added thereto, followed by phenol / chloroform / isoamyl alcohol treatment. Acetate (pH 5.2) was added and 2.5 times ethanol was added to precipitate. The synthesized cDNA was purified through Sephacryl S-400 column and quantitated cDNA was ligation of 25ng of cDNA and 10 pmol of EcoRI adapter for 18 hours at 14 ℃ and inactivated at 70 ℃ for 10 minutes, 40 μl of 0.1 mM ATP, 70 mM Tris-HCl (pH7.8), 10 mM MgCl ₂ , 5 mM DTT, and 10 units T4 polynucleotide kinase were treated. Sephacryl S-400 spin columns were performed to remove non-ligation adaptors.

Expression vector preparation

Expression vector was produced as in the schematic diagram of FIG. That is, the carrier of CyMV cDNA was used as pBluescript KS, and incubated for 12 hours in 50ml LB medium containing pBluescript KS containing ampicillin (50µg / ml), followed by centrifugation and removal of the medium, followed by alkaline lysis method. Plasmid was extracted. Pellet was resuspended by adding 1 ml of ice-cold Solution I (50 mM glucose, 25 mM Tris-Cl (pH 8.0), 10 mM EDTA (pH 8.0) and vortexing to the solution II (0.2 N NaOH, 2 ml of 1% SDS) was shaken to the side and soaked in ice for 5 minutes, then ice-cold Solution III (60 ml of 5M potassium acetate, 11.5 ml of glacial acetic acid, 28.5 ml of H ₂ O) 1.5 After adding ㎖, shake to the side and soak for 5 minutes on ice, centrifuge and transfer the supernatant to a new tube using miracloth or cheesecloth, add the same volume of phenol / chloroform / isoamyl alcohol and mix well. After centrifugation for 10 minutes, the aqueous layer was taken, and 0.1 volume of 3 M sodium acetate (pH5.2) was added thereto, and 2 volumes of ethanol were mixed and mixed well. After 1 hour storage at -20 ° C, centrifugation was performed at 12,000xg for 20 minutes. The supernatant was discarded and washed once with 70% ethanol and lyophilized. After dissolving in E-buffer, transfer to microfuge tube, process RNase, add the same volume of phenol / chloroform / isoamyl alcohol, mix well, and centrifuge at 12,000xg for 5 minutes to get an aqueous layer. Add alcohol, mix well, and centrifuge again to get an aqueous layer. Add 0.1 times volume of 3M sodium acetate (pH 5.2), add 2 times volume of ethanol, mix well, and store at -20 ℃ for 1 hour. The supernatant was discarded by centrifugation at 12,000 × g for 1 minute, washed once with 70% ethanol, and lyophilized, and the plasmids were cut with EcoRI, cut onto a band of 0.8% agarose, and then electroelution using a dialysis bag. After elution, add the same volume of butanol to purify DNA, and then centrifuge. Add the same volume of phenol / chloroform / isoamyl alcohol, mix well, and centrifuge at 12,000xg for 5 minutes. After taking the same volume of chloroform / isoamyl alcohol was mixed well and centrifuged again to take an aqueous layer. Add 0.1 times volume of 3M sodium acetate (pH 5.2), add 2 times volume of ethanol, mix well, store for 1 hour at -20 ℃, centrifuge at 12,000xg for 20 minutes, discard supernatant, and wash once with 70% ethanol. Next, lyophilized, 10X CIP dephosphorylation buffer and CIP (calf intestinal alkaline phosphatase) were added to pBluescript KS cut with EcoRI, and reacted at 37 ° C. for 30 minutes to dephosphorylation pGreenscript KS cut with EcoRI, followed by 5 mM EDTA (pH 8.0) Heated at 75 ° C. for 10 minutes. Into the same volume of phenol / chloroform / isoamyl alcohol was mixed well and centrifuged for 5 minutes at 12,000xg. Take an aqueous layer, add the same volume of chloroform / isoamyl alcohol, mix and centrifuge at 12,000xg for 5 minutes, transfer the aqueous layer to a new tube, and add 0.1 times volume of 3 M sodium acetate and 2.5 times volume of ethanol. Then stored at -20 ° C for 1-2 hours. After centrifugation at 12,000xg for 20 minutes at 4 ℃, the pellet was washed with 70% ethanol, lyophilized and dissolved in TE buffer (pH 8.0).

Ligation of CyMV cDNA with Vectors

10μl (10ng) of CyMV cDNA with ligation of EcoRI adapter, 7μl (50ng) of EcoRI-cut pBluescript KS treated with CIP, add 2μl of 10X T4 DNA ligase buffer and 1μl of T4 DNA ligase (10units) The reaction was carried out at ˜16 ° C. for 8 hours.

Competent cell preparation and transformation

E. coli JM109 was inoculated in 30 ml LB broth and incubated for 12 hours. 500 μl was inoculated into 30 ml LB broth to incubate until 0D ₆₀₀ reached nearly 0.5. The cultured cells were centrifuged at 5,000 rpm for 10 minutes, discarded, and resuspended in _1,2 ml of ice-cold 0.1 M CaCl ₂ , and 200 µl were dispensed into microfuge tubes. 10 μl of the ligation mixture of cDNA and pBluescript KS was put into the prepared competent cell, soaked in ice for 30 minutes, heated at 42 ° C. for 90 seconds, and soaked in ice for 1-2 minutes. After adding 800 μl of LB medium, incubated at 37 ° C. for 1 hour, 50 μl / ml of ampicillin, 16 mg of 50 mg / ml X-gal and 4 μl of 200 mg / ml IPTG were added to the LB plate. Smeared.

CyMV cDNA Clone Selection

In order to confirm that CyMV cDNA was inserted into pBluescript KS, the transformed cells were plated in LB solid medium containing X-gal and IPTG. After 12 hours of incubation at 37 ° C, blue / white screening was performed and the white colony was inoculated in 3 ml LB liquid medium to which ampicillin (50 µg / ml) was added. The plasmids were extracted, cut with EcoRI, and electrophoresed on agarose gel. . One of these, 400bp pCyMV ₃ was selected as a probe and Southern blot was performed using ECL. Agarose gel was treated with denaturation solution (0.5 N NaOH, 1.5 M NaCl) for 45 minutes, neutralization solution (1 M Tris pH7.4, 1.5 M NaCl) for 30 minutes, 15 minutes twice, and capillary in nylon membrne. The membrane was prepared by transfer and fixation with ultraviolet rays. Each of the membranes was immersed in 2 x SSC and then transferred to another container. Then, the hybridization buffer (including the blocking agent) was added and prehybridized at 42 ° C for 1 hour and 30 minutes. The probe to be used is boiled for 5 minutes, denaturated, soaked in ice for 5 minutes, spin down, added with the same amount of DNA labeling reagent as the probe, mixed well, and then mixed with the same amount of glutaraldehyde solution. After reacting for 10 minutes at 37 ℃. The probe thus prepared was placed in a hybridization buffer and then overnight at 42 ° C., and then washed twice at 55 ° C. with primer washing buffer for 10 minutes each and twice with secondary washing buffer for 5 minutes at room temperature. The water in the membrane was removed, a detection reagent was added, the X-ray film was exposed, and the film was developed. In addition, Northern blot was performed using the clone, pCyMV ₁ as a probe, to confirm that the longest clone was synthesized from CyMV. 1 illustrates the synthesis of pCyMV ₁ and its structure as described above. The expression vector was about 1.6 kb in size.

Hereinafter, the process of determining the nucleotide sequence by subcloning the clone selected in the present invention again.

Step 3: Subcloning and Sequence Determination

Subcloning

Clones selected through Southern and Northern blot, ie pCyMV ₁ , were cut with EcoRI and electrophoresed on agarose gel, followed by elution of only the inserts. These restriction enzymes were used as BamHI, EcoRV, HindIII, MspI, PstI, PvuII, SacI Cleavage with SmaI, XbaI. Cut into well-cut MspI, MspI + PvuII, PstI and then treated with T4 DNA polymerase to make blunt end, electrophoresis on agarose gel, cut the corresponding parts and elution each, then lipped into CIP-treated SmaI-cut pBluescript KS And transformed into E. coli JM109. The prepared restriction map is shown in FIG. 2. The transformed cells were plated in LB solid medium containing X-gal and IPTG, incubated at 37 ° C for 12 hours, and 5 white colonies were added to 3 ml LB liquid medium containing 5 ampicillin (50 μg / ml). After inoculation and culture, the plasmid was extracted, digested with HindIII + SmaI, and appropriate subclones were selected.

Sequencing

Seven subclones were prepared according to the restriction map, and the base sequence of pCyMV ₁ was determined using forward and reverse primers (FIG. 3).

In other words, the nucleotide sequence of CyMV cDNA was determined by sequenase version 2.0 of USB according to Sanger's method. 2-3 μg of the plasmid was denaturated with 0.1 M volume of 2 M NaOH / 2 mM EDTA at 37 ° C. for 30 minutes, and then 0.1 M volume of 3 M sodium acetate (pH5.2) was added, followed by 2.5 times volume of ethanol. Next, after 1 hour storage at -20 ℃ centrifuged at 12,000xg for 20 minutes to discard the supernatant, washed twice with 70% ethanol and lyophilized. 1 μl of pUC-M13 sequencing primer was added thereto, 2 μl of reaction buffer (200 mM Tris-HCl / pH 7.5, 100 mM MgCl ₂ , 250 mm NaCl) and 7 μl of H ₂ O were added at 65 ° C. for 2 minutes. Chilled slowly over minutes. A, T, G, and C were labeled in four different tubes and 2.5 μl of each termination mix (80 μM each of dGTP, dATP, dCTP, 8 μM ddNTP, 50 mM NaCl) was added. 2 µl of the labeling mix was added to the annealed tube and labeled at room temperature for 2-5 minutes. First, 3.5 µl of each of the termination mix was warmed to 37 ° C, reacted for 5 minutes, and then 4.5 µl (95% formamide, 20 mM EDTA stop solution). , 0.05% Bromophenol Blue, 0.05% Xylene cyanol FF) was added, and then treated at 75 ° C.-85 ° C. for 10 minutes, collected by centrifugation, and 3 μl of each lane of the sequcening gel was added. 6% acryamide was used for sequencing gel. After electrophoresis, it was fixed with 10% methanol and 10% acetic acid, dried and exposed to X-ray film for 1-2 days.

As with other potexviruses, the nucleotide sequence of CyMV showed that the envelope protein gene of CyMV was located on the 3 'side and the polyadenylation of the 3' side (Sonenber et al., 1978). The envelope protein was translated from the 660bp nucleotide. Could know. Comparing the nucleotide sequence of CyMV with that of CyMV reported by Chia et al., It showed about 94% similarity in nucleotide level, and most showed differences between T and C, and A and G. In the Potexvirus, the hexanucleotide motif, 5'-ACUUAA, is conserved in the 3'non-coding region, which is underlined in Figure 3, and in the potexvirus this motif is mostly about 30-40 bp above the poly (A) CyMV. In the case of, we can see that this motif exists at 59th from poly (A). This part was considered to be involved in the generation of negative-strand genomic RNA by acting as a cis-acting element involved in template recognition.

Fourth Step: Preparation of Plant Expression Vector

Insertion of CyMV Envelope Protein Gene into Plant Expression Vector

pCyMV1 was cut with PstI + EcoRI and treated with T4 DNA Polymerase to make blunt end, followed by electrophoresis on 0.8% gel to cut and elution the band containing the envelope protein gene. After ligation of the SmaI-cut pGEM-7Z (+) vector, transformation to E. coli JM109, 12 white colonies were selected, DNA was prepared, and cut with EcoRI and BamHI to confirm the envelope protein gene and insertion.

In addition, the DNA was cut into BamHI and PvuII, and the insertion direction of the envelope protein gene was confirmed, and the sequencing was confirmed again. As a result, these clones were classified into those inserted in the forward direction and antisense in the T7 promotor. Named pCyCPS and pCyCPA, respectively (Fig. 4).

Meanwhile, pMBPI was cultured in LB liquid medium to which kanamycin (50 ㎍ / mL) was added to prepare DNA, which was then cut into XbaI, treated with CIP, and electrophoresed to elution.

Likewise, pCyCPS was cut with XbaI to elution the band containing CyMV envelope protein gene, ligation to XbaI-cut pMBPI, and transformed into E. coli JM109 to obtain 15 clones. The transfromated cells were plated in LB solid medium containing kanamycin (50 μg / mL) and four transformant clones were selected. The clones were cultured in 3 mL LB liquid medium containing kanamycin (50 μg / mL). After preparation, cut each with XbaI and HindIII + KpnI to confirm the insertion and orientation of the envelope protein gene. The clones obtained here were named pMBPCPS and pMBPCPA, respectively (FIG. 5).

When these clones were cut with HindIII + KpnI, two major bands except for the vector part emerged when inserted in the forward direction, and only one main band appeared when inserted in the reverse direction, so each clone was cut with HindIII + KpnI and then 1.2% agarose. As a result of confirming the insertion direction of each clone by electrophoresis on the gel, as shown in Figure 6, the clones of lanes 2-3, which had only one band of 1.8 kb, had the envelope protein gene of CyMV reversely inserted to the 35S promoter. The clones of lanes 4-5 with two bands, 0.8kb and 1.0kb, were inserted forward. Each of the clones thus obtained was named pMBPCPS / J in the forward direction and pMBPCPA / J in the reverse direction. Here, the transformed E. coli strain was deposited with the accession number KCTC 8838P to the Biotechnology Research Institute on October 17, 1997.

Transformation to Agrobacterium tumefaciens LBA4404

In addition, pMBPCPS / J and pMBPCPA / J were transformed into Agrobacterium tumefaciens LBA4404 to prepare plant expression vectors for developing virus resistant plants. DNAs were extracted from the resulting transformant, cut with HindIII + KpnI, followed by electrophoresis, and Southern blot was performed by probe of the envelope protein gene of CyMV.

That is, Agrobacterium tumefaciens LBA4404 was incubated with YEP medium at 28 ° C. until the OD ₆₀₀ reached almost 0.5, and then centrifuged to collect the cells, and then the medium was removed and 10 ml of 0.15 M NaCl was added and resuspended. The supernatant was removed by centrifugation again, and the pellet was resuspended in 1 ml of ice-cold 20 mM CaCl ₂ , followed by aliquoting of 200 μl. In each tube, pMBPCPS / J and pMBPCPA / J were placed on ice for 30 minutes and then soaked in liquid nitrogen for 1 minute and then soaked at 37 ° C. for 5 minutes. Then, 800 μl of YEP medium was added to each tube and incubated at 28 ° C. for 3 hours, and then plated on YEP solid medium containing kanamycin (50 μg / μl), followed by incubation at 28 ° C. for 2-3 days. The transformants were then cultured in a 3 ml YEP liquid medium containing kanamycin (50 μg / μl), followed by extracting DNA, cutting the DNA with HindIII + KpnI, followed by electrophoresis (FIG. 7a), and probe the envelope protein gene of CyMV. Southern blot was performed (Fig. 7b).

Plant expression vectors pMBPCPS and pMBPCPA DNA were inserted into Agrobacterium tumefaciens LBA4404 and these clones were named pMBPCPS / L (sense) and pMBPCPA / L (antisense), respectively. As shown in FIG. 7B, Southern blot of the envelope protein gene of CyMV was probed. As a result, hybridization reaction occurred at 0.8 kb for pMBPCPS / L and 1.8 kb for pMBPCPA / L. Here, the transformed A. tumefaciens strain was deposited with the accession number KCTC 8839P to the Biotechnology Research Institute on October 17, 1997.

As can be seen through the process description and examples described above, the present invention has the effect of developing an orchid plant resistant to CyMV when transformed to the host of CyMV using the plant expression vectors pMBPCPS and pMBPCPA.

In addition, the present invention is to improve the quality of Korean orchid plants and internationally if applied to orchid plants after studying the degree of resistance to CyMV by preferentially transforming tobacco, which transforms well using the plant expression vector of the present invention. It is a very useful invention from genetic engineering, horticultural industry, and orchid plant export industry.

Claims (4)

CyMV envelope protein gene with the following nucleotide sequence was introduced into SmaI-cut pGEM-7Z (+) vector, E. coli JM109 was modified, and the selected clones pCyCPS and pCyCPA were introduced into XbaI-cut pMBⅠ. Plasmid vector pMBPCPS in which the CyMV envelope protein gene was inserted forward among clones obtained by modifying coli JM109.

E. coli JM109 (Accession No. KCTC 8838P) transformed with the plasmid vector according to claim 1. Orchid Plant Mosaic Virus (CyMV) Orchid plant expression vector inserted pCyMV ₁ with coat protein. After introducing the CyMV envelope protein gene having the nucleotide sequence of claim 1 into the SmaI-cut pGEM-7Z (+) vector, E. coli JM109 was modified, and the selected clones, pCyCPS and pCyCPA, were introduced into XbaI-cut pMBⅠ. The plasmid vector pMBPCPA in which the CyMV envelope protein gene was reversely inserted among the clones obtained by modifying coli JM109.

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