KR100250902B1 - Method of producing plants having resistance in cymv - Google Patents

Abstract

PURPOSE: Provided is a method for manufacturing plants resistant against CyMV by inserting an expression vector containing genes of CyMV envelop protein into a targeted plant. CONSTITUTION: A method for manufacturing plants resistant against CyMV is comprised of the next steps of: i) separating the gene of CyMV envelop protein from Nicotiana occidentalis; ii) inserting the gene into Sma l-cut pGEM-7Z(+) vector; iii) transforming E. coli JM109 with the vector to obtain clones pCyCPS and pCyCPA; iv) inserting the clones into Xba l-cut pMB I; v) transforming Agrobacterium tumefaciens LBA 4404 with the vector; vi) checking the insertion direction of envelop proteins (pMBPCPS is in sense direction and pMBPCPA in antisense direction); and vii) transforming targeted plants with the vectors.

Description

How to produce plants resistant to CyMV

The present invention relates to the production of plants such as virus-resistant orchids, tobacco through viral envelope protein transduction. More specifically, the present invention provides a method of producing a viral resistant transgenic plant by preparing a recombinant gene by purely separating coat protein genes of cymbidium mosaic virus (CyMV) and then introducing them into plant cells. It is about.

Since eggs are the main object of ornamentals and stems, the economic value of pests is more serious than any other crops because once the insect pests are damaged, the commodity value falls and must be disposed of. In the damage caused by pests, the most important factor that degrades egg quality is damage caused by various viral diseases, and the viral disease caused by CyMV in egg plants is the most serious in the world. Symptoms of the virus include decreased flower production, increased malformation, decreased flower quality, and loss of flower buds. In severe cases, the plant dies. In addition, CyMV virus is based on egg cultivation, which is widely cultivated, and it is known to exhibit symptoms that lower the product value of eggs (Zettler et. Al., Plant Disease, 74, 621-625, 1990).

Conventional research has been conducted to reduce the degree of virus infection and its damage to eggs, but the effect is still insignificant. The reason for this is that the mechanism of infection of the virus has not been clearly identified and typical life is not shown. Also, chemical control of microorganisms such as insects, fungi and bacteria is difficult for the virus. Recently, however, research on the viral infection mechanism has been actively conducted by the rapid development of molecular biology and genetic engineering techniques (Fraser, Annu. Rev., 28, 179-200, 1990). Development has made it possible to transform not only dicots but also monocots and other crops using electroporation and bombardment (Songstad et. Al., Plant Cell.Org.Cult, 40, 1-15, 1995). In order to develop virus-resistant plants by genetic engineering techniques, methods of inserting envelope protein genes into plants, using satellite RNA, and obtaining antiviral plants by inhibiting the expression of viral genes using antisense RNA Are known (Scolthof et. Al., Plant Physiol. 102, 7-12, 1993).

CyMV is a virus named by Jensen (Phytopath, 41, 491-494, 1951), which has a curved rod shape, 500 nm in length, 18 nm in width, 7 days retention limit, and 65-75 C heat resistance. Group. In particular, the RNA genome size of CyMV is 6.8 kb, the size of the envelope protein is 25-26 kDa, and the base sequence of the CyMV envelope protein gene was determined by Ryu et al. (Gene, 156, 303-304, 1995). Attempts have been made to produce transformants by expressing in plants.

Accordingly, an object of the present invention is to prepare a cDNA library from CyMV viral RNA, and insert the CyMV envelope protein gene isolated therefrom into a suitable vector carrying genes to plant cells, thereby providing a recombinant vector suitable for transforming plant cells. There is.

Still another object of the present invention is to provide a method for producing a plant transformed with the recombinant vector to generate a CyMV envelope protein gene in a cell, thereby transforming a plant having a low CyMV resistance into a plant resistant to the virus. To provide.

In order to achieve the above object, the present inventors propagate the CyMV envelope protein gene to Nicotiana occidentalis, which is the growth host of CyMV, isolate CyMV viral RNA, prepare a cDNA library from 1 μg viral RNA, and then prepare a primer specific for CyMV CyMV envelope protein gene was purified purely. The isolated CyMV envelope protein genes were cloned and their entire sequences were determined by Sanger's dideoxy chain termination (Sanger, Science, 214, 1205-1210, 1981).

In order to express the isolated CyMV envelope protein gene in plants, fragments of a binary vector pMBP1 were linked to complete vectors pMBPCPS and pMBPCPA for use in plant transformation.

In addition, pMBPCPS and pMBPCPA were introduced into Agrobacterium tumefaciens LBA 4404 to express CyMV envelope protein genes in plants, and plant cells were transformed with Agrobacterium. The vector was deposited with the accession number (KCTC 8839P) at the Biotechnology Research Institute on October 17, 1997. Transformed plant cells induce a suit in a shoot induced selection medium containing 100 μg / ml kanamycin and 300 μg / ml carbenicillin. The induced chute was placed on a selection medium to induce roots, and the root-derived plants were transferred to pots to purify and continued to grow. In order to test whether the CyMV envelope protein gene introduced into the plant cell was expressed, PCR was performed to confirm that the CyMV envelope protein gene was introduced into the genome of the plant. In addition, the Northern blot method was used to determine whether the gene was transcription. Finally, CyMV was inoculated into the transformed plants to assay the resistance to the virus.

Hereinafter, the present invention will be described in detail with reference to examples. The following examples are merely provided to illustrate the present invention, the scope of the present invention is not limited thereto.

Figure 1 shows the complete nucleotide sequence of the CyMV envelope protein gene purely isolated from the cDNA library,

2 is a diagram illustrating a sequence of introducing pMBPCPS and pMBPCPA into Agrobacterium tumefaciens LBA 4404 to express CyMV envelope protein genes in plants.

Figure 3 shows the transformation process of tobacco cells using the expression vector and pMBPCPS and pMBPCPA in order,

4 shows the results of PCR using NPTII specific primers from transformed tobacco,

Figure 5 shows the results of performing Northern (Northern) analysis by separating the total RNA isolated from the tobacco to which the CyMV envelope protein gene was introduced,

Figure 6 shows the viral test 15 days after inoculation of the transformed tobacco (top leaf has no symptoms)

Figure 7 shows the results of virus assay showing that the secondary lobe is infected with the virus 5 weeks after inoculation of untransformed tobacco.

Figure 8 shows the results of virus assay of the secondary lobe 5 weeks after inoculation of the transformed tobacco.

Example 1

CDNA synthesis and cloning from CyMV viral RNA

CyMV viral RNA was isolated by PEG (polyethylene glycol) precipitation and centrifugation, and viral RNA was purified by two phenol extraction and phenol / chloroform (1: 1) extraction.

Purified 1 μg of viral RNA was isolated and purified mRNA using oligo (dT) cellulose column, and 5 μg of mRMA was isolated to synthesize cDNA using ZAP-cDNA synthesis kit (Stratagene, U.K.). After attaching an EcoRI adapter to the synthesized cDNA, cDNA synthesized with Sephacryl S-400 spun column was fractionated according to size. Fractionated cDNA was combined with Uni-Zap XR vector (Stratagene, U.K.) and then subjected to in-vitro packaging using a packaging extract.

Example 2

Sequence determination of CyMV cDNA

The sequencing of CyMV cDNA was performed according to the method of Amersham thermo sequenase cycle sequencing kit. In order to isolate the CyMV gene, fragments of the amplified PCR reaction products were labeled using a DIG-Labeling & Detection kit (Boehringer Mannheim, Germany) and used as probes. First, phages were infected with E. coli XL1-Blue to form a plaque of about 6 × 10 ⁵ pfu in an 82-mm petri dish, and incubated at 37 ° C. for 12 hours. Screening was performed. Only white colonies were obtained from screening and in vivo excision was performed using R408 helper phage to transfer phagemids from recombinant Uni-ZAP XR phages to E. coli XL1-blue. Each of the generated colonies was selected to separate plasmids by alkaline hemolysis (alkaline detergent) method, and double-cut by EcoRI and KpnI to select clones with amarathin gene.

The clones isolated were subcloned into pBlueScript SK (-) vectors to determine sequencing. Selected clones were digested with PstI and subjected to self ligation to transform into transformable XL1-Blue, followed by LB medium (10 g / l Bactotrypton, 5 g / l) containing 50 μg / ml ampicillin. Colonies generated in Yeast exrtact, 5 g / L NaCl, pH 7.5) were selected, plasmids were isolated by alkaline hemolysis, digested with HindIII + SmaI, and appropriate subclones were selected. The DNA obtained from each colony was purified and the base sequence of the CyMV cDNA insert was determined by using a sequinase (USB) as a dideoxy chain termination method using SK promoter primer and T7 promoter primer.

As shown in FIG. 1, as a result of determining the nucleotide sequence of the CyMV cDNA insert of pMBP1, the total size is about 1.5kbp, and one of the structural genes for synthesizing the CyMV envelope protein is one open reading frame consisting of 660bp. As with other potexviruses, the envelope protein gene of CyMV is located on the 3 'side and the 3' polyadenylation site. In FIG. 1, (*) represents a stop codon, and the amino acid sequence was prepared according to IUPAC nomenclature. As shown in FIG. 1, the CyMV envelope protein was composed of about 35 kDa protein consisting of 200 amino acids.

Meanwhile, comparing the nucleotide sequences of other CyMV envelope protein genes, CyMV-S (Chia et. Al., Plant Mol. Biol., 18, 1901-1099, 1992) and CyMV-K (Ryu et. Al., Gene) , 156,303-304, 1995) and nucleotide sequences showed 93% and 91% identity, respectively. These were 44 and 59 bases substituted in nucleotide sequence, respectively.

Example 3

Manipulation of binary vector pMBP1 and construction of pMBPCPS and pMBPCPA

As a transport vector for plant expression, pMBP1 was used and an expression vector in which the envelope protein of CyMV was inserted in the reverse direction and the forward direction was used (FIG. 2).

pCyMV1 was cut into 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, it was transformed into E. coli JM109, 12 white colonies were selected, DNA was prepared, and cut into EcoR I and BamH I to confirm the envelope protein gene and insertion.

In addition, the DNA was cut into BamHI and PvuII to confirm the envelope protein gene and the insertion direction, and the result was confirmed again through sequencing. As a result, the clone was classified into one inserted into the T7 promotor and the other inserted into the antisense. These were named pCyCPS and pCyCPA, respectively.

Meanwhile, pMBP 1 was cultured in LB liquid medium to which kanamycin (50 µg / ml) was added to prepare DNA, which was then cut into Xba I and subjected to CIP, followed by electrophoresis.

Likewise, pCyCPS was cut into Xba I to elution the band containing the CyMV envelope protein gene, ligation to Xba I-cut pMB I and transformed into E. coli JM109 to obtain 15 clones. The transformed 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, HindIII + KpnⅠ and confirm the insertion and the direction of insertion of the envelope protein gene. The clones obtained were named pMBPCPS and pMBPCPA, respectively.

Example 4

Agrobacterium-mediated transformation of plant cells

Plant-transformed binary vectors pMBPCPS and pMBPCPA obtained in Example 3 in which the CyMV envelope protein gene was regulated under the CaMV 35S promoter were freeze-dissolved for transformation with Agrobacterium tumefaciens LBA 4404 ( freeze-thawing) method was used. In order to select Agrobacterium tumefaciens LBA 4404 transformed with the vector, DNA was isolated by Agrobacterium plasmid quick-screen method and digested with restriction enzyme HindIII + Kpn I to confirm the transformation. (Stanton et. Al., Plant Molec.Biology Manual, Kluwer Academic Publishers, 1988).

Agrobacterium tumefaciens LBA 4404 transformed with pMBPCPS and pMBPCPA was deposited with the Accession No. (KCTC 8839P) at the Biotechnology Institute on October 17, 1997.

Nicotiana occidentalis was used for the transformation of tobacco cells. The tobacco leaf grown in the greenhouse was treated with 70% (v / v) ethanol for 30 seconds, then sterilized with 2% sodium hypochloride for 15 minutes, washed 5-6 times with sterile water, and cut into 8mm squares to transform tobacco cells. Used as. Agrobacterium tumefaciens LBA 4404 transformed with pMBPCPS and pMBPCPA was incubated for 18 hours at 28 ° C. and 200 rpm in LB medium. After incubation, the cells were recovered by centrifugation, and suspended in a cell number of about 1 to 2 x 10 ³ per ml with MS basic medium, and used for transformation of tobacco cells. Tobacco leaf slices were co-cultured in agrobacterium tumefaciens LBA 4404 suspension with pMBPCPS and pMBPCPA for 1 day, followed by 2.0 mg / l benzyl aminopurine (BAP), 100 mg / l kanmycin and 300 mg / l carbenicillin. The chute was induced in the added MS selection medium. Cultivation conditions were 25 ℃, 2500Lux, the length of the 16-hour cycle was used, the chute differentiated from leaf slices were subcultured every two weeks as in the culture of leaf slices. Transgenic tobacco re-differentiated in MS selection medium was transplanted into MS root selection medium containing 50mg / l kanamycin and 300mg / l carbenicillin to induce root formation. It was used for the next experiment. Tobacco transformation process is shown in FIG.

Example 5

PCR analysis on transformed tobacco

In order to confirm whether the envelope protein gene of CyMV was stably introduced into the genome in the tobacco transformed with the pMBPCPS and pMBPCPA vectors, the NPT II gene was amplified by PCR to confirm the transformation indirectly (FIG. 4). In order to confirm that CyMV envelope protein gene was stably introduced into the genome of the plant, genomic DNA was isolated from the potential transformant and PCR was performed. PCR was performed using 100 mM Tris-HCl, 500 mM KCl, 1.0% Triton X-100, 25 mM mgCl ₂ , 0.1 mM dNTP, 50 pmol primer, and 4 units of Tag polymerase. Primers used for PCR synthesized the nucleotide sequence specific for the NPTII gene (Back et. Al., Gene, 19, 327-326, 1982). PCR was synthesized using a DNA synthesizer (ABI, USA) and denatured with Vent ^tm DNA polymerase (NEB, USA) using a DNA thermocycler (Cetus / Perkin-Elmer, USA) (94 35 ° C., 1 min), annealing (60 ° C., 1 min) and extension (72 ° C., 2 min) were performed. As shown in Figure 4 it was confirmed that the same size of the NPT II gene amplified in plants transformed in the same manner as the pMBPCPS (fourth (a)) and pMBPCPA (fourth (b)) vector (lanes each: 3,4 ). Plants that were not transformed did not amplify the NPT II gene (lanes 2). Each lane 1 shows a pMBPCPS vector and a pMBPCPA vector, and M lane shows the size marker cut | disconnected with Lamda HindIII.

Example 6

Northern blot analysis of transformed tobacco

In order to determine the transcription level of the envelope protein gene of CyMV in tobacco transformed with pMBPCPA vector, total RNA was extracted from tobacco indirectly transformed in Example 5 and 10% (v / v) formaldehyde was extracted. After electrophoresis on 1% (v / v) agarose gel containing the RNA was transferred to the nylon membrane and hybridization was carried out using the envelope protein gene probe of CyMV synthesized by PCR. The results are shown in Figure 5, there was no band in the non-transformed control (lane N), it can be seen that only one band appears only in the transformed tobacco (lane 1,2).

Example 7

Viral Assay of Transformed Tobacco

Lyophilized CyMV diseased tissue purchased from ATCC was crushed with phosphate buffer solution, centrifuged to obtain supernatant, virus inoculated solution was inoculated to transgenic tobacco and control (leaf 5-6 leaf) It was. Bioassay 15 days after inoculation showed that the mosaic of the typical symptom of CyMV was found in the whole plant, but the transformed tobacco showed no symptoms in the upper leaf, as shown in FIG. . As a result of observing the infection of the secondary lobe 5 weeks after inoculation, as shown in Fig. 7, the control group showed no mosaicism, which is a typical symptom of CyMV, while the transformed tobacco showed no symptoms (Fig. 7). 8).

As described and demonstrated in detail above, the present invention transforms a plant cell into a CyMV coat protein gene expression vector prepared by inserting a gene encoding the CyMV coat protein gene into a vector enabling expression of a foreign gene in the plant cell. By converting and culturing, the present invention provides a method for producing a plant that produces CyMV envelope protein. Therefore, by producing the CyMV envelope protein and manufacturing a plant by the method of the present invention, various plants having strong CyMV resistance can be developed. It is a very useful invention.

Claims (3)

CyMV envelope protein gene isolated from Nicotiana occidentalis, the growth host of CyMV, was introduced into Sma I-cut pGEM-7Z (+) vector, modified with E. coli JM109, and selected clones pCyCPS and pCyCPA. Of the clones obtained from Xba I-cut pMB I and modified E. coli JM109, agrobacterium transformed with pMBPCPS with CyMV envelope protein gene forwardly inserted and pMBPCPA with CyMV envelope protein gene reversely inserted Tumefaciens LBA 4404 (Strain Deposit No. KCTC 8839P).

A method of producing a plant having resistance to CyMV by culturing a plant transformed by a freeze-dissolution method via the Agrobacterium tumefaciens LBA 4404 (Strain Accession Number KCTC 8839P) of claim 1. The method of claim 2, wherein the plant producing resistance to CyMV is Nicotiana occidentails.

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