南方水稻黑条矮缩病毒P7-2基因的克隆与原核表达

Cloning and Prokaryotic Expression of P7-2 of Southern rice black-streaked dwarf virus

【目的】为克隆获得南方水稻黑条矮缩病毒(Southern rice black-streaked dwarf virus,SRBSDV)的P7-2基因并实现其原核表达。【方法】利用RT-PCR技术从感染SRBSDV的水稻叶片中扩增出P7-2基因。利用Gateway重组技术将基因P7-2整合到原核表达载体pDEST17上,获得重组原核表达载体pDEST17-P7-2。随后将原核表达载体pDEST17-P7-2转化到原核表达菌株Rosetta中。利用IPTG诱导P7-2蛋白表达并优化其诱导条件。【结果】利用RT-PCR技术扩增得到基因P7-2,其大小为930 bp。测序结果表明获得原核表达载体pDEST17-P7-2。菌液PCR鉴定了原核表达阳性菌株。在温度为28℃、IPTG浓度为0.1 mmol/L诱导表达8 h,可获得大量大小约为42 kDa含HIS标签的融合蛋白。【结论】克隆获得了SRBSDVP7-2的基因序列,实现了该基因的原核表达并探索其最佳诱导条件,为南方水稻黑条矮缩病的田间诊断、预测预报及P7-2的功能研究奠定了基础。

[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.