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以采自广东省水稻瘤矮病的典型病株为材料,通过RT-PCR法扩增和克隆了水稻瘤矮病毒(Rice gall dwarf virus,RGDV)S11组分核苷酸序列全长。序列分析结果表明,RGDV广东分离物S11(登录号EF532326)核苷酸序列全长1168bp,含有一个1068bp的开放阅读框,编码一条355个氨基酸的多肽,推测分子量约40kD,与泰国分离物基因结构基本一致,其核苷酸与氨基酸序列相似性分别为94.3%和98.8%。将广东分离物S11基因克隆至pBV221温敏表达载体上,在大肠杆菌DH5α中实现了高效表达。以诱导蛋白为抗原免疫家兔获得了抗血清。利用ELISA法测定抗血清的效价为1∶4096,Western blot分析结果表明,抗血清能与S11蛋白发生特异血清学反应。这可为进一步研究S11蛋白的结构和功能,揭示其抑制基因沉默的作用机制提供参考。
The full-length nucleotide sequence of the S11 component of rice gall dwarf virus (RGDV) was amplified and cloned by RT-PCR from the typical diseased plants collected from Guangdong Province. Sequence analysis showed that the RGDV Guangdong isolate S11 (accession number EF532326) was 1168 bp in length and contained a 1068 bp open reading frame encoding a polypeptide of 355 amino acids with a deduced molecular weight of about 40 kD. Basically the same, the nucleotide and amino acid sequence similarity were 94.3% and 98.8%. The S11 gene of Guangdong isolate was cloned into the pBV221 thermosensitive expression vector and highly expressed in E. coli DH5α. Antisera were obtained by immunizing rabbits with the induced protein as antigen. The titer of antiserum was 1:4096 by ELISA. Western blot analysis showed that antiserum could specifically react with S11 protein. This may provide a reference for further studying the structure and function of S11 protein and revealing its mechanism of inhibiting gene silencing.