甲基营养菌MP688甲醇脱氢酶基因mpq1818的敲除及功能研究  

Knockout and Characterization of mpq1818 Gene of Methylovorus sp.MP688

作　　者：李大攀 葛欣[2] 魏静远 熊向华[2] 汪建华[2] 杨秀萍[1] 张惟材[2] 

机构地区：[1]首都师范大学生命科学学院,北京100048 [2]军事医学科学院生物工程研究所,北京100071

出　　处：《生物技术通讯》2014年第5期632-635,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:研究甲醇脱氢酶基因mpq1818在甲基营养菌MP688生长代谢中的作用。方法:利用同源重组原理构建中间为庆大霉素抗性基因Gmr、两侧mpq1818基因上下游序列同源的敲除载体pAK0-up-Gmr-down,接合转移导入MP688,通过庆大霉素抗性和组合PCR方法筛选基因敲除菌,并检测其生长、甲醇脱氢酶活性、甲醇利用及吡咯喹啉醌(PQQ)生物合成能力等方面的差异。结果:抗性和PCR验证显示mpq1818缺失株构建成功;与野生菌相比,缺失株的甲醇脱氢酶活力及利用甲醇的能力降低,而且菌株的生长和PQQ产量也有显著下降。结论:基因mpq1818的缺失影响菌株前期生长与PQQ合成。Objective： To study the function of the mpq1818 gene from Methylovorus sp. MP688. Methods： The deletion mutant of mpq1818 was constructed by homologous recombination. The mutant strain was verified by PCR and anehored-PCR. We compared the differences of biological characteristics such as growth rate, the utilization of methanol, the gross activity of methanol dehydrogenase（MDH） and the level of pyrroloquinoline quinine（PQQ） production between the mpq1818 knockout mutant and parent strain MP688. Results： The gene of mpq1818 was successfully knocked out. The results showed that the volume of methanol utilization and the total activities of MDH were significantly lower than that of parent strain MP688. And then, it resulted in a slow growth rate and a drop of PQQ secretion. Conclusion： The mpq1818 gene may influence the growth rate and the biosynthesis of PQQ in the prophase of Methylovorus sp. MP688.

关 键 词：甲基营养菌 甲醇脱氢酶 基因敲除 吡咯喹啉醌 

分 类 号：Q78[生物学—分子生物学]