海南番木瓜环斑病毒全长cDNA克隆及其侵染性克隆构建

The Isolation and Infectious Clone Construction of Complete Genomic Sequence of Papaya ringspot virus from Hainan Island China

为了从分子水平上解析番木瓜环斑病毒（Papaya ringspot virus,PRSV）的致病机理,寻找广谱、有效的抗病毒新策略,培育具有应用前景的抗病新品种,利用RT-PCR和快速cDNA末端扩增技术（RACE）对引起番木瓜环斑病毒病的PRSV海南海口分离物（PRSV-HN2）的基因组cDNA进行了全序列测定。PRSV-HN2全长cDNA包含10 326 nt（不包括3′端的polyA,Gen Bank登录号为KF791028）,能编码3 346个氨基酸。对PRSV-HN2基因组核苷酸序列及其推导的氨基酸序列进行了结构和功能分析,结果表明,其与其它23个PRSV分离物的核酸序列相似度达81%-91%,氨基酸相似度为88%-94%。利用酵母同源重组系统成功构建其侵染性克隆载体并通过农杆菌转化与侵染,最终获得其侵染性克隆。这为在分子水平上研究PRSV遗传变异、侵染及致病机理奠定理论基础。

In order to understand the pathogenesis mechanism of papaya ringspot virus at the molecular level, and to find a more broad-spectrum and effective antiviral strategy, therefore to cultivate new varieties with resistant promising, the complete genomie sequence of papaya ringspot virus isolated from Hainan island in China was cloned and determined by using RT-PCR and rapid-amplification of eDNA ends （RACE） techniques （GenBank accession number： KF791028）. The full-length cDNA contains 10 326 nt （not including the 3＇ end of the poly （A））, a total of 3 346 amino acids are encoded, which was Compared with other PRSV isolates, the nucleotide sequence similarity was 81% to 91%, also the amino acid similarity was 88% to 94%. The full-length cDNA was cloned into plant expression vector, then the infectious clone was obtained by Yeast homologous recombination system and further transformed into Agrobacterium tumefaciens. The research of genetic variation PRSV, infection and pathogenesis at the molecular level will be benefit from this study.