水稻瘤矮病毒S11基因沉默抑制子的原核表达及抗血清制备

Prokaryotic expression and antiserum preparation of a RNA silencing suppressor encoded by Rice gall dwarf virus segment 11

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 of nucleotide sequence of Rice gall dwarf virus（RGDV） segment 11（S11） was amplified with RT-PCR from the diseased samples collecting from Guangdong Province.The results showed that S11 had 1 168 nt encoding a polypeptide of 355 amino acids with a Mr of 40 kD.Sequence comparison showed that S11 of RGDV Guangdong isolate had the same genome organization with the Thai isolate,and shared 94.3% nucleotide and 98.8% amino acid sequence identities,respectively.The S11 cDNA was cloned to pBV221 and the protein was highly expressed in Escherichia coli DH5α.The expressed protein（Pns11） was used to immunize the rabbit,and the antiserum against Pns11 with titer above 1∶4 096 measured by ELISA was obtained.Western blot analysis indicated that the antiserum could serologically react with Pns11 expressed in the transgenic rice plants with genetic transformation of RGDV S11.These work will lay foundation for further research on structural feature and functional mechanism of the RGDV Pns11 protein.