云南烟草花叶病毒54KD基因的分离、克隆及序列分析  

Cloning and Sequencing of A 54KD Gene from Tobacco Mosaic Virus

作　　者：王芳[1] 鄢波[1] 俞立 陈利[1] 张仲凯[1] 黄兴奇[1] 

机构地区：[1]云南省农科院生物技术研究所

出　　处：《西南农业学报》1998年第4期10-17,共8页Southwest China Journal of Agricultural Sciences

基　　金：云南省自然科学基金

摘　　要：从云南省弥勒县采集典型病株接种蔓陀萝，植斑分离，经免疫电镜（ＩＥＭ）鉴定为ＴＭＶ（Ｔｏｂａｃｃｏｍｏｓａｉｃｖｉｒｕｓ），在红花大金元品种中以ＴＭＶ普通株作对照进行致病力测定，确定为强毒株系。以烟草花叶病毒ＲＮＡ云南强毒株系为模板，根据国内外研究结果自行设计、合成寡核苷酸引物，通过ＲＴ—ＰＣＲ体外扩增，Ｅ．ｃｏｌｉ菌株ＤＨ５α克隆，获得了云南烟草５４ＫＤ（ＴＭＶ复制酶）全基因片段。顺序分析表明：该基因含１４２５个核苷酸，编码４７５个氨基酸，与国外发表的Ｕ１株系比较，ＤＮＡ同源率为９９．０％，氨基酸同源率为９８．９％，与国内普通株比较，ＤＮＡ同源率为９９．１％，氨基酸同源率为９８．９％。According to the nucleotide sequences of 54KD gene that reported in China and abroad,we designed and synthesized a pair of primer.RNA was isolated from tobacco mosaic virus (Yunnan strain),1 45KD DNA fragment was obtained with RT PCR technique.After purification,the DNA fragement was inserted into pGEM T vector system directly and transformed into E.coli DH 5α cells.The white colony named pVHZ2,which was screened with X gal,was chosen for further analysis of restriction endonuclease map.The gene was subcloned in order to analysis of sequence,the full of DNA sequence of the gene was determined and compared with the sequences reported before.We found that it is highly homologous both in DNA and in deduced amino acid sequences.

关 键 词：烟草花叶病毒 54KD基因 PCR 克隆 序列分析 

分 类 号：S435.72[农业科学—农业昆虫与害虫防治]