华北农学报2017，Vol.32Issue(1)：68-72,5.DOI:10.7668/hbnxb.2017.01.011
灰葡萄孢BcKMO基因的原核表达分析
Prokaryotic Expression Analysis of BcKMO Gene from Botrytis cinerea
摘要
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.关键词
灰葡萄孢/BcKMO/原核表达/纯化/PCRKey words
Botrytis cinerea/BcKMO/Prokaryotic expressin/Purification/PCR分类
农业科技引用本文复制引用
王敏,刘媛媛,周帆,姜婷婷,郑旭,张靖,时翠平,邢继红,董金皋..灰葡萄孢BcKMO基因的原核表达分析[J].华北农学报,2017,32(1):68-72,5.基金项目
河北省高等学校科学技术研究项目(ZD2016001) （ZD2016001）
