草莓镶脉病毒(SVBV)ORF Ⅵ基因的克隆及蛋白特性分析

Cloning and Protein Property Analysis of ORF Ⅵ Gene of Strawberry Vein Banding Virus(SVBV)

用CTAB法从感染草莓镶脉病毒(strawberry vein banding virus,SVBV)的草莓叶片中提取总DNA,设计特异性引物扩增SVBV ORF Ⅵ基因的全长片段,进行克隆并测序.序列分析表明SVBV ORF Ⅵ基因完整序列全长1 557bp,编码518个氨基酸.将其与SVBV美国分离物以及花椰菜花叶病毒属其他成员的ORF Ⅵ基因相比较,结果表明SVBV中国分离物ORF Ⅵ基因与SVBV美国分离物ORF Ⅵ基因核苷酸序列相似性最高,达84.5%;而与花椰菜花叶病毒属其他成员的ORF Ⅵ基因序列相似性均较低,仅为23.7%～27.9%.构建SVBV及其同属其他成员ORF Ⅵ基因的系统关系树,结果显示SVBV中国分离物与SVBV美国分离物单独形成1个分支,说明来源于草莓的2个SVBV亲缘关系最近,而与其同属其他成员的亲缘关系均较远.生物信息学分析表明SVBVORF Ⅵ基因表达的P6蛋白序列含有多种蛋白激酶磷酸化位点,其二级结构N端是1个α-螺旋富集区.该研究将为进一步研究P6蛋白的功能提供理论依据.

Total DNA was extracted from strawberry leaves infected with strawberry vein banding virus（SVBV） by CTAB method.Using a specific primer,the fragment of SVBV ORF Ⅵ gene was amplified,cloned and sequenced.Sequence analysis showed that the full length of SVBV ORF Ⅵ gene was 1557 nts,encoding 518 amino acids.Comparison of SVBV ORF Ⅵ gene with the American isolate of SVBV and the other members of Caulimovirus showed that the ORF Ⅵ gene of the Chinese isolate of SVBV shared high sequence similarity（84.5%） with that of the American isolate of SVBV and had relatively low nucleotide sequence similarity（23.7%~27.9%） with the other members of Caulimovirus.A phylogenetic tree based on alignment of ORF Ⅵ nucleotide sequences of SVBV and the other members of Caulimovirus was constructed,which indicated that the Chinese isolate of SVBV and the American isolate of SVBV were clustered into a separate branch,that the two SVBV derived from strawberry had the closest relationship and that they had relatively distant relationship with the other members of Caulimovirus.The data obtained by bioinformatics analysis demonstrated that the P6 protein expressed by ORF Ⅵ gene of SVBV contained many kinds of protein kinase phosphorylation sites and the secondary structure of P6 contained an α-helix-rich region at its N-terminus.