KR20000018072A - Nucleotide and amino acid sequences of full-length coat protein gene of cucumber green mottle mosaic virus (CGMMV) Y strain - Google Patents

Abstract

PURPOSE: Nucleotide sequence of coat protein gene of CGMMV type Y is useful for the specific diagnosis of viral disease. CONSTITUTION: Nucleotide and amino acid sequences(drawing 3) of coat protein of CGMMV type Y, which are cloned into pGEMT-Easy vector. CGMMV is isolated from cucumber leaves, which are infected with virus and total RNAs of CGMMV are purified by phenol extraction method. The nucleotide sequence of PCR product, which is cloned into pGEMT-Easy vector is determined and amino acid sequence is sequenced therefrom

Description

Nucleotide and amino acid sequences of full-length coat protein gene of cucumber green mottle mosaic virus (CGMMV) Y strain}

Among the fruits and vegetables, watermelon is an important crop in Korea, and is grown in large quantities in Korea, Asia, Europe, and the United States. However, watermelons and melons are very economically damaging due to various viruses, and there are no pesticides to control these viruses. Therefore, the international trend of making and using virus-resistant transgenic plants for the control of viruses. Is in. Cucumbers include cucumber green mottle mosaic virus (CGMMV), zucchini yellow mosaic virus (ZYMV) and cucumber mosaic virus (CMV). . In particular, when CGMMV develops in watermelon, not only the severe symptoms of the leaves but also the fleshy part of the watermelon becomes colorless watermelon, which has no commercial value. CGMMV is a major economically damaging virus from the mid 1980s to the present, centering on the major watermelon cultivation sites in Korea. Therefore, in the present invention, genetically engineered CGMMV envelope protein genes that can be directly used for genetically engineered virus-resistant plants for the purpose of controlling CGMMV, and can be used for artificial antibody production and plant expression virus vector development. Cloning was performed and their nucleotide and amino acid sequences were determined.

The present invention relates to the envelope protein nucleotide sequence and amino acid sequence of the Cucumber green mottle mosaic virus (CGMMV) Y strain. The present invention relates to synthesizing and cloning cDNA for coat protein genes in genomic genes of the CGMMV-Y family to determine the nucleotide sequence, from which the amino acid sequence of the coat protein is determined.

1 is a severe mortar and mosaic symptom in watermelon by CGMMV,

2 is a CGMMV-Y strain purified virus electron micrograph,

Figure 3 is a 524 bp CGMMV-Y line coat protein gene DNA product by RT-PCR (arrow 1 in Figure A, M: 1kb plus DNA ladder), the product of Figure 1 in the plasmid vector (pGEMT-Easy ) Cloned clone clone (clone name: pCGY-CP) was treated with EcoR I restriction enzyme and then inserted CGMMV-Y envelope protein gene (arrow 2 in Figure B, M: 1kb plus DNA ladder) It is a picture confirmed by electrophoresis.

1 gaagagtcca gttctgtttc ttttgaagat gacttacaat ccgatcacac ctagcaaact

61 tattgcgttt agtgcttctt atgttcccgt caggacttta cttaattttc tagttgcttc

121 acaaggtacc gctttccaga ctcaagcggg aagagattct ttccgcgagt ccctgtctgc

181 gttaccctcg tctgtcgtag atattaattc tagattccca gatgcgggtt tttacgcttt

241 cctcaacggt cctgtgttga ggcctatctt cgtttcgctt ctcagctcca cggatacgcg

301 taatagggtc attgaggttg tagatcctag caatcctacg actgctgagt cgcttaacgc

361 tgtaaagcgt actgatgacg cgtctacggc cgctagggtt gagatagata atttaataga

421 gtctatttct aagggttttg atgtttacga tagggcttca tttgaagccg cgttttcggt

481 agtctggtca gaggctacca cctcgaaagc ttagtttcga gggt

1 MTYNPITPSK LIAFSASYVP VRTLLNFLVA SQGTAFQTQA GRDSFRESLS

51 ALPSSVVDIN SRFPDAGFYA FLNGPVLRPI FVSLLSSTDT RNRVIEVVDP

101 SNPTTAESLN AVKRTDDAST AARVEIDNLI ESISKGFDVY DRASFEAAFS

151 VVWSEATTSK A

Example 1 Virus Isolation

In June 1998, diseased watermelons were collected, showing severe mosaics and mottles in Naju (Figure 1), from which the virus was biologically isolated.

Example 2 Virus Purification and Gene Isolation

The virus was artificially inoculated with healthy cucumbers in the cotyledon state to multiply the virus, and the virus was purified from the material. Virus genes were purified from the purified virus by phenol extraction.

Example 3 Cloning of Envelope Protein Genes

Using the purified viral RNA as a template, a primer specific for the envelope protein was prepared, and the target gene was amplified in a large amount by RT-PCR, and the recombinant gene was prepared by TA cloning on the pGEMT-Easy (Promega) vector. The selected recombinant gene was digested with EcoR I restriction enzyme using EcoR I sites at both ends of the cloning site to confirm the size of the inserted gene (Fig. 3), and DNA sequences were determined using these. The amino acid sequence was determined based on the determined base sequence, and the cloned gene was identified as a CGMMV envelope protein through genetic analysis.

In the present invention, genetically engineered cloning protein genes of CGMMV, which can be directly utilized for genetically engineered virus-resistant plants for the purpose of controlling CGMMV, and can be used for artificial antibody production and plant expression virus vector development. Their nucleotide and amino acid sequences were determined.

Claims (3)

Envelope Protein DNA Sequences and Amino Acid Sequences of CGMMV-Y Lines and Their Functional Equivalents Cloning method of CGMMV-Y line coat protein gene and preparation of plant transformation vector and functional equivalents Protein production using Escherichia coli expression using CGMMV-Y line coat protein, antibody production using it and development of virus resistant plant through expression in higher plants and functional equivalents