Prokaryotic Expression Analysis of BcKMO Gene from Botrytis cinerea

doi:10.7668/hbnxb.2017.01.011

Abstract

Abstract: The aim of this study is prokaryotic expression analysis of BcKMO gene from Botrytis cinerea and obtain the purified BcKMO protein.The BcKMO gene was amplified by RT-PCR technology using the cDNA of the Botrytis cinerea wild type BC22,cloned into the pMD19-T vector and sequenced.The results of sequencing showed that the BcKMO gene sequence was right.The pMD19-T-BcKMO and pGEX4T-1 plasmids were digested using restriction enzyme.The BcKMO gene segments were collected and cloned into the pGEX4T-1 vector.The results of restriction enzyme digestion and sequencing showed that the vector pGEX4T-1-BcKMO-GST was successfully constructed.The vector pGEX4T-1-BcKMO-GST was transformed into E.coli BL21 strain.The results of IPTG inducement indicated that the pGEX4T-1- BcKMO -GST was successfully expressed in E.coli BL21 strain,with the molecular weight 71 kDa.The optimal conditions of the prokaryotic expression of BcKMO were determined as 0.2 mmol/L IPTG treatment 12 h.Western Blot results showed that the GST antibody could specifically bound to purified pGEX4T-1-BcKMO-GST fusion protein,suggesting that the expression of BcKMO gene was successfully in vitro.

Key words: Botrytis cinerea, BcKMO, Prokaryotic expressin, Purification, PCR

CLC Number:

S431.03
Q78

WANG Min, LIU Yuanyuan, ZHOU Fan, JIANG Tingting, ZHENG Xu, ZHANG Jing, SHI Cuiping, XING Jihong, DONG Jingao. Prokaryotic Expression Analysis of BcKMO Gene from Botrytis cinerea[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2017, 32(1): 68-72. doi: 10.7668/hbnxb.2017.01.011.

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