南方水稻黑条矮缩病毒P7-2基因的克隆与原核表达OA

[Objective]To establish a method for rapid detection of Southern rice black-streaked dwarf virus(SRBSDV)and study the function of P7-2 gene,it is necessary to prepare specific antiserum of P7-2.[Method]The P7-2 gene was amplified by RT-PCR from the rice leaves infected with SRBSDV.The purified PCR fragment of P7-2 gene was subcloned into the prokaryotic expression vector pDEST17 to obtain the recombinant prokaryotic expression vector pDEST17-P7-2 by Gateway recombinant technology.Then the recombinant vector was transformed into E.coli Rosetta cells,which was induced by IPTG to express the protein and optimize the induced expression conditions.[Result]Sequence of the 930 bp SRBSDV P7-2 was amplified by RT PCR.The sequencing results showed that the prokaryotic expression vector pDEST17-P7-2 was obtained.The positive prokaryotic expression of E.coli Rosetta strains was identified by PCR.A large number of 42 kDa HIS6-tag fusion protein was obtained with the induction of 0.1 mmol/L IPTG from E.coli Rosetta cells for 8 h when the temperature was 28℃.[Conclusion]SRBSDV P7-2 gene was obtained and its protein was induced successfully under the optimal conditions,which laid a foundation for the detection and prediction of SRBSDV in the field as well as for the function study of SRBSDV P7-2.