甲基营养菌甲醇脱氢酶基因的克隆及表达

目的:从甲基营养菌MP681中扩增甲醇脱氢酶(MDH)基因,在大肠杆菌中表达并检测其活性,同时在MP681中考察该基因对吡咯喹啉醌(PQQ)产生的影响。方法:根据MP681基因组序列设计引物,PCR扩增靶基因mdh,构建表达载体,考察活性,利用接合转移转化至MP681,考察PQQ的合成。结果:扩增得到甲基营养菌MP681甲醇脱氢酶基因,在大肠杆菌中的表达产物能够催化甲醇脱氢;将携带mdh基因的质粒转入MP681后,PQQ产量略有提高。结论:获得编码MDH的基因,该基因能够在大肠杆菌中表达,且表达产物具有生物活性;甲醇脱氢酶基因表达对宿主菌的PQQ合成可能有一定影响。

Cloning and Expression of Methanol Dehydrogenase Gene of a Methylotrophic Bacteria Strain

Objective： To amplify the methanol dehydrogenase（MDH） gene from methylotrophic bacteria stain MP681,express and confirm its activity in E.coli,and investigate its effect on pyrroloquinoline quinone（PQQ） gener-ation in MP681.Methods： To amplify the target gene mdh by PCR with primers based on MP681 genome se-quences,construct expression vectors,and test the activity,transform into MP681 through conjugation and detect the generation of PQQ.Results： Methanol dehydrogenase gene was amplified from MP681 and expressed in E.coli.The target product was able to catalyze the dehydrogenation of methanol,and the yield of PQQ of the transfor-mants with the plasmid carrying mdh gene increased slightly.Conclusion： The gene encoding MDH was obtained and the MDH was expressed in E.coli in biological active form.The expression of methanol dehydrogenase gene may influence the biosynthesis of PQQ in the host.