瓜类褪绿黄化病毒山东分离物全基因组序列扩增及分析

为明确分离自山东省寿光市甜瓜上的瓜类褪绿黄化病毒(cucurbit chlorotic yellows virus,CCYV)分离物的全基因组序列信息和遗传变异情况,利用毛形病毒属Crinivirus简并引物进行RTPCR检测,利用RACE技术结合RT-PCR方法克隆CCYV山东分离物2条RNA链的全基因组序列,通过与GenBank中其它地区CCYV分离物的全长序列进行比对分析其同源性,并基于CP基因序列构建系统进化树分析其遗传变异情况。结果表明,山东分离物经RT-PCR检测和测序后确定为CCYV。CCYV山东分离物与其它CCYV分离物的RNA1链和RNA2链的全基因组序列一致性的平均值分别为99.82%和99.88%,且2条链的5′末端均比较保守,没有碱基突变的情况发生;RNA1链3′末端存在2个碱基变异,RNA2链3′末端存在1个碱基变异。CCYV不同地区分离物主要分为3个簇群,其中山东分离物和中国其它地区分离物、日本分离物、苏丹分离物、黎巴嫩分离物和塞浦路斯分离物聚类在一起。研究表明CCYV基因组序列比较保守,该病毒的分化可能与地理来源存在一定的相关性。

Amplification and analysis of whole genome sequence of cucurbit chlorotic yellows virus from Shandong Province, China

To clarify the whole-genome sequence and genetic variation of cucurbit chlorotic yellows virus (CCYV) isolated from melon in Shouguang City, Shandong Province, degenerate primers of the genus Crinivirus were used for RT-PCR detection, and the whole-genome sequences of two RNA strands of CCYV Shandong isolate were obtained by RACE and RT-PCR cloning. The similarity of the wholegenome sequence between Shandong isolate and isolates of CCYV from other regions in GenBank was compared. The genetic variation was analyzed by constructing the phylogenetic tree based on the coat protein (CP) gene sequences. The results showed that the isolate from Shandong was identified as CCYV by RT-PCR detection and sequencing, and the average sequence identity of RNA1 and RNA2 among Shandong isolate and other isolates were 99.82% and 99.88%, respectively. The 5'' ends of the two strands of Shandong isolate were both conservative and no base mutation occurred. Furthermore, there were two base variations at the 3'' end of the RNA1 strand, and one base variation at the 3'' end of the RNA2 strand. The isolates from different geographical origins of CCYV were mainly divided into three clusters. The isolate from Shandong was clustered with isolates from other areas of China, Japanese isolates, Sudan isolate, Lebanese isolates and Cyprus isolates. This study indicated that the genomic sequence of CCYV was relatively conserved and that differentiation of the virus might be related to geographical occurrence.