葡萄A病毒外壳蛋白基因克隆及分子变异分析

为获得葡萄A病毒(Grapevine virus A,GVA)外壳蛋白(CP)基因,并为开展GVA-CP介导的抗病毒转基因研究提供基础,本研究从葡萄休眠枝条中扩增获得12个GVA分离物的CP基因序列,全长597nt,编码198个氨基酸。所得分离物间核苷酸和氨基酸序列同源性分别为83.9%~99.8%和92.4%~99.5%,与GenBank中12个GVA CP序列比较,核苷酸和氨基酸序列同源性分别为71.0%~93.1%和81.8%~98.0%。系统进化分析表明,本研究12个分离物CP序列与已报道的GVA Ⅰ组亲缘关系最近。种群变异分析表明,GVA种群具有多种变异体类型,并且多个样品存在不同变异体类型的复合侵染。但绝大多数变异体克隆聚集在GVA Ⅰ组分支,有60%的克隆序列间同源性较高,并且聚集在同一个次级分支Ⅰ Aa,表明其为该GVA种群的优势序列。克隆的GVA CP基因以及优势序列群体的分析为葡萄抗病毒转基因研究奠定了基础。

Cloning and Molecular Variation Analysis of Coat Protein Gene of Grapevine virus A

In order to obtain the coat protein (CP) gene of Grapevine virus A (GVA) and provide a basis for GVA-CP-mediated antiviral transgenic studies, we cloned 12 complete sequences of CP gene of GVA by amplification from grapevine dormant cuttings. Each complete sequence of CP gene of the 12 isolates was 597 nts long and encodes 198 amino acids. These sequences shared 83.9%~99.8% nt identity and 92.4%~99.5% amino acid identity with each other, and they shared 71.0%~93.1% nt sequence identity and 81.8%~98.0% amino acid identity with 12 sequences in GeneBank. Phylogenetic analysis showed that the CP sequences in this study shared relatively high homologies with the GVA-group Ⅰ reported previously. Population variation analysis indicated that the GVA population contained a number of variant types, and several samples were infected with more than one GVA variants; however, most of the variants clustered with the GVA-group Ⅰ isolates. 60% of the variants shared high sequence identities and clustered on the same secondary branch of phylogenetic tree known as ⅠAa, indicating that these variants were dominant in the GVA population. Cloning of the GVA CP gene and analysis of the dominant sequences laid the foundation for the grapevine anti-virus transgenic research.