云南马铃薯A病毒分布及其外壳蛋白分子变异分析

Distribution and Molecular Variation of Coat Protein of Potato Virus A from Yunnan

为研究云南马铃薯A病毒(PVA)分布及其外壳蛋白的分子变异,2007年至2011年分别从云南滇西北、滇东北、滇中及滇南马铃薯种植区采集381份马铃薯样品,进行马铃薯A病毒DAS-ELISA检测,结果表明,PVA阳性样品共43份,其中滇西北22份,滇中17份,滇东北4份,滇南未检出。对带有PVA的部分样品进行电镜负染色观察,电镜下观察到长约750 nm、直径约15 nm的线状粒子。根据已报道的PVA外壳蛋白基因(cp基因)序列设计合成特异性引物,以带毒样品总RNA为模板,RT-PCR扩增得到807 bp目的片段,克隆测序并对其推导的外壳蛋白(CP)氨基酸序列进行同源性分析,并比较分析了不同地区PVA分离物CP氨基酸序列分子变异。结果表明,云南不同地区PVA分离物与GenBank中登录的部分PVA分离物CP氨基酸序列同源性为93.3%～100%,依据不同PVA分离物CP氨基酸序列构建系统进化树,云南PVA分离物与中国福建分离物(AF483279)亲缘关系较近,形成一个进化簇。PVA不同分离物CP氨基酸序列分子变异分析表明,不同分离物CP氨基酸变异位点主要分布在氨基酸序列的N端。

A survey was conducted in the potato-growing areas in northwest,central, northeast and south Yunnan from 2007 to 2011 to determine the occurrence and distribution of potato virus diseases.381 potato samples were collected and detected by DAS-ELISA.The results showed that 43,including 22 from northwest Yunnan,17 from central Yunnan,4 from northeast Yunnan,were infected by Potato virus A（PVA）. The filamentous and flexuous particles were observed from the sap of diseased potato leaves under the electron microscopy.Total RNAs of 22 ELISA positive samples were extracted and amplified by RT-PCR with the specific primers for the coat protein gene of PVA.The DNA products of 807 bp were amplified,cloned and sequenced. Sequence analysis revealed that cp gene of PVA isolates from Yunnan shared 93.3 %-100 % identities at the amino acid level with those of other PVA isolates. Phylogenetic tree based on amino acid sequences of PVA coat protein revealed that PVA Yunnan isolates had the close relationship with Fujian isolates.The molecular variation analysis of amino acid sequences of the different PVA isolates indicated that variation sites were located in N terminal of amino acid sequence.