利用PCR介导的基因置换技术构建阿维链霉菌bkdF^-突变株  

Construction of Streptomyces avermitilis bkdF^- Mutants Using PCR-mediated Gene Replacement Method

作　　者：熊伟[1] 梁运祥[1] 朱浩君[1] 闵勇[1] 吕和平[2] 郑应华[2] 

机构地区：[1]华中农业大学农业微生物国家重点实验室,武汉430070 [2]北京生物医药研究所,北京100091

出　　处：《农业生物技术学报》2005年第5期672-678,共7页Journal of Agricultural Biotechnology

摘　　要：PCR介导的基因置换技术是一种利用λ噬菌体Red重组系统在微生物中进行基因中断的新方法。实验利用该方法快速构建基因中断载体,并成功地置换了阿维链霉菌(Streptomyces avermitilis)bkdF基因。先合成1对长度分别为58和59nt的引物,其5'端的39nt序列分别与bkdF基因两侧同源,3'端则分别与安普霉素抗性标记盒(aac(3)Ⅳ+oriT)两侧序列一致。以该引物扩增的PC R产物电转化能表达λRed重组酶且含有目标质粒的大肠杆菌(Escherichia.coli)菌株B W25113/pIJ790/pX W1224,获得了阳性重组质粒pX W1230。将该质粒中的大小为4.0kb B glⅡ目的片段插入基因置换载体pH Z1351,再接合转移至阿维链霉菌B JBM9903,筛选得到表型为ApraRThioS的接合子。PCR验证并对发酵产物进行H PLC和MS分析,证实该接合子为bkdF-突变株。PCR mediated gene replacement method is a novel way of gene knockout in bacteria which is based on λRed system. By using the method, a gene replacement vector was rapidly constructed and then bkdF gene of Streptomyces avermitilis was successfully replaced. Firstly, a pair of 58 and 59 nt primers were prepared, which had at 5＇ end 39 nt matching the flanking sequence of bkdF, and the 3＇ sequence matching the right or left end of the apramycin resistant cassettes （aac （3） Ⅳ＋ oriT）. The linear DNA PCR-amplified by these primers was electrotransformed to the strain BW25113/pU790/pXW 1224 which expressed λRed enzyme and contained target plasmid, then several positive recombined plasmids （pXW1230） were acquired. The 4.0 kb Bgl Ⅱ target fragment from pXW 1230 was inserted into pHZ 1351, and subseqnently introduced into S. avermitilis BJBM9903 by conjugal transfer. After PCR and HPLC and MS analysis of fermentation product, the screened Apra^RThio^S conjugant was confirmed to be the bkdF mutant.

关 键 词：PCR介导的基因置换 RED重组 阿维链霉菌 支链α酮酸脱氢酶基因(bkdF) 基因中断 

分 类 号：Q78[生物学—分子生物学] Q939.13