江苏省葫芦科作物西瓜花叶病毒的分子鉴定和序列分析

Molecular identification and sequence analysis for watermelon mosaic virus in Jiangsu

[目的]对江苏各地具典型褪绿花叶症状的多种葫芦科作物是否为西瓜花叶病毒（Watermelon mosaic virus,WMV）侵染进行分子鉴定,明确该病毒在江苏的遗传分化.[方法]提取样品总RNA,利用西瓜花叶病毒的cp基因特异性引物进行RT-PCR扩增,克隆测序,应用DNASTAR和Clustal X软件进行序列分析.[结果]来自南瓜、瓠瓜、西瓜和西葫芦的11个分离物的RNA模板中均检测到1200 bp左右的片段;序列测定表明该片段含1154个核苷酸,包含完整的编码281个氨基酸的WMV-cp基因.序列比对结果表明,11个分离物和来自不同地区的17个分离物WMV-cp基因核苷酸和推导的编码CP蛋白氨基酸序列同源性分别为90.5％～100.0％和92.9％～100.0％.[结论]WMV的遗传变异与地缘相关性较强而与寄主相关性较弱.江苏分离物WMV-CP氨基酸均具有蚜传株系的特征结构域DAG,暗示其田间流行与蚜虫有关.

[Objective]Molecular identification was conducted to identify whether varieties of cucurbit crops with typi- cal chlorisis mosaic symptoms from Jiangsu were infected by watermelon mosaic virus （WMV）. The genetic differentiation of WMV in Jiangsu was also defined. [Method]Total RNA was extracted from the infected isolates and the coat protein （cp） gene of WMV was amplified by RT-PCR with the specific primers. The DNA products were cloned and sequenced, and sequence analysis was conducted by DNAStar and Clustalx software. [Resuh]A fragment with the size of about 1200 bp was detected from 11 isolates including pumpkin, gourd, watermelon and zucchi. Sequence analysis indicated that the nu- cleotide fragment contained 1154 bp, including complete cp gene and encoded 281 amino acids. By DNAStar and Clustalx software, the obtained sequences were compared with 17 reported WMV-cp sequences from different regions. The similari- ties of nucleotide and deduced amino acid sequences among these isolates were ranged from 90.7% to 100.0% and 93.2% to 100.0%, respectively. [Conclusion]The phylogenetic tree of nucleotide and comparison of amino acid analysis indicated that its genetic diversity was related to geological distribution with little difference from the host origin. Jiangsu isolates contained the motif sequence DAG associated with aphid transmission.