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应用反转录-PCR技术合成并扩增了水稻矮缩病毒(RDV)中国福建分离物基因组第七号片段(S7)cDNA,将PCR产物克隆在载体 pBluescript SK(+)上,进行了亚克隆和序列分析。结果表明克隆片段全长1578bp,含有一个1518bp长的开放阅读框架(ORF),编码一个由506个氨基酸组成的理论分子量为56000的蛋白。该片段与RDV日本分离物基因组的相应片段相比,在核苷酸及氨基酸水平上的同源率分别为93.2％和94.1％。该片段编码的56000蛋白与同属的伤瘤病毒(WTV)基因组第七号片段编码的57000蛋白相比,在靠近N端的区域(aa61～aa141)有较高的氨基酸序列同源性。将RDV S7 cDNA克隆到原核表达载体 pBV221上,通过温度诱导在大肠杆菌中得到表达。表达产物占菌体可溶性总蛋白的13.73％。对表达产物进行了 Western blotting分析。本工作为进一步纯化RDV S7编码蛋白、分析其功能和转化水稻打下了良好基础。
The seventh fragment (S7) cDNA of rice dwarf virus (RDV) Fujian isolate was synthesized and amplified by reverse transcription-PCR. The PCR product was cloned into vector pBluescript SK (+) and subcloned And sequence analysis. The results showed that the cloned fragment was 1578bp in length and contained a 1518bp long open reading frame (ORF) encoding a protein with a theoretical molecular weight of 56000 consisting of 506 amino acids. The fragment shares 93.2% and 94.1% nucleotide and amino acid identity, respectively, with the corresponding fragment of the RDV Japan isolate genome. This fragment encodes a 56000 protein that has higher amino acid sequence homology to the 57000 protein encoded by the seventh fragment of the same genomic virus (WTV) genome near the N-terminal region (aa61-aa141). The RDV S7 cDNA was cloned into the prokaryotic expression vector pBV221 and expressed in E. coli by temperature induction. The expression product accounted for 13.73% of the total bacterial soluble total protein. The expression product was analyzed by Western blotting. This work laid a good foundation for further purification of RDV S7 encoded protein, analysis of its function and transformation of rice.