生命科学研究2017，Vol.21Issue(3)：208-212,256,6.DOI:10.16605/j.cnki.1007-7847.2017.03.004
一个深黄被孢霉苹果酸脱氢酶基因的克隆与表达
Cloning and Expression of a Novel Malate Dehydrogenase Gene from Mortierella isabellina
摘要
Abstract
A pair of gene-specific primers was designed based on the RNA-seq data,and a full-length cDNA fragment of 1 017 bp,designated MIMDH2,was obtained from Mortierella isabellina strain M6-22 by PCR amplification using cDNA as template.Sequence analysis showed that it comprised an open reading frame encoding 338 amino acids.The deduced amino acid sequence shared the highest identity of 71.26％ with that of Aspergillus fumigates,and it also contained the conserved coenzyme and substrate binding sites,as well as catalytic active sites.The cDNA sequence was further subcloned into expression vector pET32a (+)to generate recombinant plasmid pET32a-MIMDH2,which was subsequently transformed into Escherichia coli BL21 for inducing expression.The expressed protein was purified by Ni-NTA affinity chromatography.Enzymatic activity analysis demonstrated that the specific activity of the recombinant protein was 271.33 U/mg.All these results demonstrated that the amplified cDNA fragment MIMDH2 is a novel and potential malate dehydrogenase gene and its expressed protein has the catalytic activity of malate dehydrogenase.关键词
深黄被孢霉/苹果酸脱氢酶(MDH)/基因克隆/序列分析/原核表达Key words
Mortierella isabellina/malate dehydrogenase (MDH)/gene cloning/sequence analysis/prokaryotic expression分类
生物科学引用本文复制引用
崔锦锦,季秀玲,林连兵,魏云林,张琦..一个深黄被孢霉苹果酸脱氢酶基因的克隆与表达[J].生命科学研究,2017,21(3):208-212,256,6.基金项目
国家自然科学基金资助项目(31160016,31660454) （31160016,31660454）
云南省应用基础研究基金资助项目(KKSA201126005) （KKSA201126005）
教育部回国人员科研启动基金(KKQA201226003) （KKQA201226003）
