灰葡萄孢BcKMO基因的原核表达分析  

Prokaryotic Expression Analysis of BcKMO Gene from Botrytis cinerea

作　　者：王敏[1] 刘媛媛[1] 周帆[1] 姜婷婷[1] 郑旭[1] 张靖[1] 时翠平[1] 邢继红[1] 董金皋[1] 

机构地区：[1]河北农业大学真菌毒素与植物分子病理学实验室,河北保定071001

出　　处：《华北农学报》2017年第1期68-72,共5页Acta Agriculturae Boreali-Sinica

基　　金：河北省高等学校科学技术研究项目(ZD2016001)

摘　　要：为构建灰葡萄孢犬尿氨酸单加氧酶(Bc KMO)基因的原核表达载体并进行高效表达,获得纯化的Bc KMO蛋白。以灰葡萄孢野生型BC22为试材,通过反转录PCR扩增Bc KMO基因,回收Bc KMO基因片段克隆到p MD19-T载体中,测序正确后,酶切p MD19-T-Bc KMO和带有GST标签蛋白的p GEX4T-1质粒,将目的片段进行连接,构建Bc KMO基因的原核表达载体p GEX4T-1-Bc KMO-GST。经酶切和测序鉴定正确后,将构建好的原核载体转化大肠杆菌BL21。经IPTG诱导,在大肠杆菌BL21菌株中成功表达了与GST标签蛋白融合的Bc KMO蛋白,大小约71 k Da。SDS-PAGE分析表明,该蛋白在0.2 mmol/L IPTG诱导12 h时高效表达;Western Blot结果发现目的蛋白能与GST特异性抗体起特异性反应,表明Bc KMO基因的体外诱导表达成功。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-! 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 con- structed. The vector pGEX4T-1-BcKMO-GST was transformed into E. coli BL21 strain. The results of IPTG induce- ment 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 PCR 

分 类 号：S431.03[农业科学—农业昆虫与害虫防治] Q78[农业科学—植物保护]