水稻草矮病毒基因组vRNA3 NS3基因的克隆、序列分析及原核表达

摘要: 根据RGSV(rice grassy stunt virus )-IR分离物的RNA3序列设计引物，采用RT-PCR技术扩增出RGSV-SX分离物的NS3基因，并进行序列测定及原核表达。结果表明，NS3基因由588个核苷酸组成，编码22.9 kD蛋白。构建了可在E. coli DH5α中表达的质粒pGTNS3，经IPTG诱导，SDS-PAGE分离纯化得到分子量为49.0 kD的GST-NS3融合蛋白，并制备抗血清。应用Western blot 分析寄主水稻(Oryza sativa)和介体昆虫体内NS3基因表达产物，仅在感病水稻植株中检测到NS3蛋白，而在提纯病毒、介体昆虫体内则未检测到。

Cloning, Sequence Analysis and Prokaryotic Expression of the vRNA3 NS3 Gene in Rice Grassy Stunt Virus Shaxian Isolate

Abstract: Rice grassy stunt virus (RGSV) is classified as a member of Tenuivirus . The filamentous particles of RGSV are ribonucleo-proteins (RNPs), which are composed of a single nucleocapsid (NC) protein and genomic ssRNA segment. RGSV caused rice yield to lose a lot in South and Southeast Asia during the 1970's. It also occurred in South of China, such as Fujian, Taiwan, Guangdong, Guangxi and Hainan Provinces. The whole sequence of RGSV genome was determined in 1998 by Toriyama et al, and the result revealed that RGSV has six genomic RNA segments, RNA segments 1, 2, 5 and 6 correspond to RNA 1-4 of other tenuiviruses. And all six RNA segments had an ambisense coding strategy, which is unique not only in genus Tenuivirus but also in plant viruses. The unique RNA segment 3 contains an ORF on each of vRNA and vcRNA. However, the functions of RNA3 are still unknown. The potential function of vRNA3 was studied by cloning, sequencing and expression of RGSV vRNA3 NS3 gene. Based on the known RNA sequence of RGSV-IR isolate, the cDNA of the vRNA3 NS3 gene was obtained by RT-PCR with genomic RNAs of RGSV-SX isolate as template. The cDNA was then cloned and sequenced. The results showed that NS3 gene was composed of 588 nt and the sequence identities were 99.1% and 96.2% at the nucleotide level, 98.4% and 96.4% at the amino acid level, comparing with those of RGSV- IR and SC published isolates. The 22.9 kD protein encoded by RGSV-SX vRNA3 showed 33.0% identity between 80 amino acids compared with the 21.6 kD protein encoded by vRNA5 of RGSV, and no other significant matches were found in GenBank. The similarity between the 22.9 kD protein and the 21.6 kD protein suggested that vRNA3 of RGSV might have sort of recombination with other RGSV RNA segments and/or unknown RNAs. Using recombinant plasmids containing vRNA3 NS3 gene and vector of pGEX-2T, we constructed prokaryotic expression plasmids pGTNS3 which produced 49.0 kD fusion protein of GST-NS3 in E. coli, prepared the antiserum against the fusion protein and used it for the detection of RGSV by Western blot. The encoded protein was detected only in infected rice (Oryza sativa ), nothing in purified virus and viruliferous planthopper vectors. These results would provide more information for the further study on the functions of the vRNA3 NS3 gene and its encoded protein.