烟草花叶病毒54 kD基因的cDNA克隆及序列分析

cDNA CLONING AND SEQUENCING OF GENE ENCODING 54 kD PROTEIN OF TOBACCO MOSAIC VIRUS

以国内普通株系的烟草花叶病毒(TMV)RNA为模板,人工合成的寡聚核苷酸为引物,合成了cDNA并克隆到Bluescript载体上。通过限制性内切酶图谱分析和DNA全序列测定,证明克隆了完整的烟草花叶病毒的54kD蛋白基因。测定的cDNA序列与国外发表的TMV U_1株系比较,DNA有很高的同源率,编码的氨基酸序列完全相同。并将该基因转入烟草中,获得了在卡那霉素选择培养基上生长的愈伤组织。

The RNA genome was purified from tobacco mosaic virus (TMV) infecting Chinese tobacco plants. The cDNA of the gene encoding 54 kD protein of TMV was synthesized using the RNA genome as template, and a piece of artificial oligonucleotide as primer. The cDNA was cloned into pBluescript, generating pRC19, and identified by restriction endonuclease digestion. Determination of the nucleotide sequence of the cDNA fragment shows that the gene is 1422 bp in length and encodes a protein of 474 amino acids. The gene is highly homologous in DNA sequence to that of TMV U_1 strain. The deduced amino acid sequence of the 54 kD proteins of both strains are identical. The 54 kD gene was transformed to tobacco plants and the transgenic calli growing on selective medium were obtained.