生命科学研究2017，Vol.21Issue(6)：482-487,6.DOI:10.16605/j.cnki.1007-7847.2017.06.003
红冬孢酵母苹果酸酶基因RKME1的克隆与表达
Cloning and Expression of Malic Enzyme Gene RKME1 in Rhodosporidium kratochvilovae
摘要
Abstract
Malic enzyme (ME) is a widely distributed enzyme involved in different metabolic pathways in prokarvotic and eukaryotic microorganisms.It catalyzes the oxidative decarboxylation of L-malate to yield pyruvate,CO2 and NADPH in the presence of a bivalent cation (Mg2+ or Mn2+).The results of the previous analysis showed that the YM25235 strain had the expression of two ME iso-enzyme genes RKME1 and RKME2,and the mRNA transcription level of RKME1 gene was significantly improved at 15 ℃.Thus,the sequence of RKME1,which encoded a total of 540 amino acids,with an open reading frame of 1 623 bp,was obtained by PCR with YM25235 cDNA as template.Sequence analvsis showed that there are four conserved domains of malic enzyme (Ⅰ～Ⅳ) in the sequence,and homology modeling showed the tertiary structure of the sequence had high similarity with that of the malate enzyme in Ascaris suum.In order to verify its function,RKME1 was further inserted into vector pET32a(+) to construct a recombinant expression plasmid pET32a-RKME1,which was then introduced into E.coli BL21 for expression.A new protein band with relative molecular mass of about 70 kD was obtained by IPTG induction.The result of enzymatic activity analysis after purification by nickel affinity chromatography showed that recombinant expression of RKME1 protein could catalyze the dehydrogenation of malic acid to pyruvate and produce NADPH,and the cnzyme activity was more than 160 U/mg.The above results indicate that RKME1 is a novel malic enzyme gene and could lay a foundation for further study of the relationship between malic acid enzyme and growth adaptability of Rhodosporidium kratochvilovae at low temperature.关键词
红冬孢酵母/苹果酸酶/基因克隆/生物信息学/重组表达/酶活Key words
Rhodosporidium kratochvilovae/malic enzyme/clone/bioinformatics/recombinant expression/enzyme activity分类
生物科学引用本文复制引用
肖虎,熊向峰,崔锦锦,季秀玲,林连兵,魏云林,张琦..红冬孢酵母苹果酸酶基因RKME1的克隆与表达[J].生命科学研究,2017,21(6):482-487,6.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）
