云南烟草花叶病毒外壳蛋白基因的分离克隆及序列分析

Cloning and sequencing of the gene encoding coat proteinfrom tobacco mosaic virus(Yunnan strain)

从云南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；在烤烟品种红花大金元上以ＴＭＶ普通株作对照进行致病率测定，确定为强毒株系。以烟草花叶病毒（ＴＭＶ）云南强毒株系ＲＮＡ为模板，根据国外研究结果，自行设计、合成寡核苷酸为引物，通过ＰＴ－ＰＣＲ体外扩增，得到约５００ｂｐ的ＤＮＡ片段，将其克隆到Ｅ．ｃｏｌｉＤＨ５α上，并进行了序列分析。分析表明，该其因含４７７个核苷酸，编码１５９个氨基酸，与国外发表的Ｕ１株系比较，核苷酸同源率为９８．１％，氨基酸同源率为９７．５％。获得了云南烟草ＴＭＶ外壳蛋白全基因片段。

RNA was isolated from tobacco mosaic virus(Yunnan strain).According to the nucltotide sequence of coat protein that reported abroad,we designed and synthesized a pair of primer.A 053kb DNA fragment was obtained with RTPCR technique.After purification,the DNA fragment encoding coat protein was inserted into pUC18 vector system and transformed into Ecoli DH5 cells.The white clone named pVHY5 from screening with Xgal and IPTG was chosen,the full DNA sequence of the gene was determined and compared with the sequence reported abroad.We found that it is highly homologous both in DNA and in deduced amino acid sequences.The gene was cloned,and the medium vector can express in the plant.