Avermectin C5-O-甲基转移酶基因的克隆、序列分析及其基因置换  

Study on C5-O-methyltransferase gene cloning, sequencing and replacement

作　　者：朱浩君[1] 郑应华[2] 

机构地区：[1]华中农业大学生命科学院,武汉430070 [2]北京生物医药研究所,北京100091

出　　处：《中国抗生素杂志》2003年第1期1-5,59,共6页Chinese Journal of Antibiotics

摘　　要：C5 O 甲基转移酶基因 (aveD)位于阿维链霉菌 (Streptomycesavermitilis)染色体 3 4kbBamHI的片段上 ,通过构建avermectin基因组约 3 4kbBamHI片段的亚基因文库 ,用PCR的方法获得该基因的连锁基因C5 酮基还原酶基因 (aveF) ,并作为探针筛出aveD的克隆子。测序后和GenBank做同源比较 ,结果一致。构建基因置换穿梭载体 pHJ5 80 3 (pHZ13 5 8∷aveD&Amr) ,通过ET12 5 67::pUZ80 0 2属间接合转移导入S .avermitilis。放松培养后筛选出双交换重组菌株 ,并用PCR验证重组菌株的aveD已经破坏 ,将重组菌株摇瓶发酵液提取avermectin进行色谱质谱联机分析 ,发现重组菌株仅产生 4个组分 ,与出发菌株的 4个B组分完全一致。C5 O methyltransferase gene is a key gene that is involved in biosynthesis of avermectin A. C5 O methyltransferase gene was cloned from the sub library constructed by the pool of 3 4kb Bam HI DNA fragment of Streptomyces avermitilis chromosome by using C5 ketoreductase gene as the probe.The sequence analysis indicated that the cloned gene was identical to the C5 O methyltransferase gene in S.avermitilis . The gene replacement vector, pHJ5803 (apra+), was constructed and introduced into S.avermitilis by conjugation. The gene replacement strain Bjbm0006, which no longer produce the avermectin A, was obtained and confirmed.

关 键 词：AVERMECTIN C5-O-甲基转移酶 基因置换 接合转移 

分 类 号：R978.15[医药卫生—药品]