利用同源重组建立地中海拟无枝菌酸菌U32染色体的基因置换/中断系统  

Establishment of Gene Replacement/disruption System Through Homologous Recombination in Amycolatopsis mediterranei U32

作　　者：丁晓明[1] 张霓[1] 田永强[1] 姜卫红[1] 赵国屏[1] 焦瑞身[1] 

机构地区：[1]中国科学院上海生命科学研究院植物生理生态研究所,上海200032

出　　处：《生物工程学报》2002年第4期431-437,共7页Chinese Journal of Biotechnology

基　　金：国家自然科学基金 (No.39970 0 0 7);国家杰出青年基金 (No .30 12 5 0 0 2 )资助~~

摘　　要：地中海拟无枝菌酸菌U32是力复霉素SV的工业产生菌 ,其遗传操作一直是一个难题。在该菌株DNA高效电转化的基础上 ,利用同源重组的原理 ,建立了地中海拟无枝菌酸菌染色体的基因置换 中断系统。通过大肠杆菌重组质粒pDK110构建、转化及两步重组筛选 ,成功地用α 淀粉酶基因 (amy)取代了地中海拟无枝菌酸菌U32染色体上的 3 氨基 5 羟基苯甲酸合成酶基因 (ahbas)。第一步单交换和第二步双交换的频率分别是 0 5 %～ 0 7%和2 %。将质粒pDK110变性后转化可显著提高重组频率 ,在第二步筛选双交换前对单交换重组子进行电击也能够提高其双交换重组的频率。此外 ,通过转化构建的两端带同源区段的线性DNA片段及一步重组筛选 ,我们在地中海拟无枝菌酸菌U32染色体的amrD ,rifO基因中间插入了阿普拉霉素抗性基因 (apr) ,其效率约为 30～ 5 0转化子 μgDNA。A gene replacement/disruption system of Amycolatopsis mediterranei U32 was developed based on the established electroporation conditions as well as appropriate selective markers. Through two-step selection, ahbas gene in U32 was replaced by a promoterless α-amylase gene constructed on the plasmid pDK110 of E.coli. The first single-crossover and the second double-crossover frequencies were approximately 0.5%～0.7% and 2%, respectively. Denaturation of the plasmid pDK110 increased the integration frequency about 7～10 folds, while electric shock treatment of the single-crossover recombinants increased the frequency of second crossover recombination about 5 folds. Employing denatured DNA fragments containing an apramycin-resistance gene flanked with regions of the respective genes, One-step disruption of rifO and amrA genes of U32 was also achieved with an efficiency of 30～50 transformants per microgram of DNA.

关 键 词：同源重组 地中海拟无枝菌酸菌U32 染色体 基因置换/中断系统 力复霉素 

分 类 号：Q784[生物学—分子生物学]