通过转座诱变筛选甲基营养菌吡咯喹啉醌合成缺陷突变株  

Screening of PQQ-Deficient Mutants by Transposition Mutagenesis

作　　者：韩月梅[1,2] 葛欣[1] 王鹤[2] 熊向华[1] 汪建华[1] 刘党生[2] 张惟材[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100071 [2]沈阳药科大学,辽宁沈阳110016

出　　处：《生物技术通讯》2014年第5期628-631,共4页Letters in Biotechnology

基　　金：国家自然科学基金(31300083);中国博士后科学基金(2013M542443)

摘　　要：目的:对电转化等Tn5转座诱变条件进行优化,获得大量甲基营养菌MP688突变株,筛选吡咯喹啉醌(PQQ)合成缺陷突变株,并对失活基因进行鉴定。方法:通过电击方法对MP688株进行Tn5转座诱变;通过检测PQQ产量,选择不产或几乎不产PQQ的突变株,用质粒拯救的方法鉴定突变基因。结果和结论:确定了MP688株电击转化的最优条件,优化了质粒拯救法鉴定突变基因的实验方案,得到了1株PQQ合成明显降低的突变株RM16,并确定了转座子在染色体上的插入位点。Objective： To obtain a considerable amount of Tn5 transposition mutants of Methylovorus sp. MP688 and screen pyrroloquinoline quinine （PQQ） -deficient strains and identify the mutation genes, the electroporation and other conditions involved in Tn5 transposition mutagenesis were optimized. Methods： Tn5 transposition mutagenesis for MP688 were carried out by electroporation. The PQQ-deficient and the PQQ-declined mutants were chosen by measuring the fermentation broth＇s PQQ content, The mutation genes were identified by plasmid rescue. Results ＆ Conclusion： The electroporation transformation procedure was optimized and the mutation genes were identified when using plasmid rescue. And then a mutant with an obvious decline in PQQ secretion were named RM16 and the insertion site in the chromosome was successfully identified.

关 键 词：甲基营养菌 吡咯喹啉醌 Tn5转座 电击转化 突变 

分 类 号：Q78[生物学—分子生物学]