高山被孢霉Δ5去饱和酶基因的克隆及异源表达研究  

Cloning and Heterologous Expression of Δ5 Desaturase Gene from Mortiella alpina

作　　者：刘建民[1,2] 栗茂腾[1] 

机构地区：[1]华中科技大学生命科学与技术学院,湖北武汉430074 [2]河南理工大学资源环境学院,河南焦作454000

出　　处：《食品科学》2013年第15期166-170,共5页Food Science

基　　金：教育部新世纪优秀人才支持计划项目(NCET110172);国家自然科学基金青年科学基金项目(41202113);河南理工大学博士基金项目(B2011-035)

摘　　要：通过RT-RCR的方法从高山被孢霉W15菌株中扩增到完整的Δ5去饱和酶基因,其全长为1341bp,编码446个氨基酸。对推导的氨基酸序列进行分析表明,其N末端存在细胞色素b5结构域,在序列中还存在3个保守的组氨酸富集区。将该基因连接到表达载体pPIC3.5K上,转化毕赤酵母细胞,利用G418筛选高拷贝数的转化子。以双高γ-亚麻酸为底物,在甲醇诱导的条件下,Δ5去饱和酶可催化底物脱氢生成花生四烯酸,其含量占总脂肪酸含量的2.85%。说明克隆的Δ5去饱和酶基因能在毕赤酵母中进行功能性的表达。Arachidonic acid, an essential fatty acid for humans, plays important physiological functions in the intelligence development and vision development of infants. In this study, a complete Δ5 desaturase gene was cloned from Mortierella alpina W15 by RT-PCR amplification. The gene with 1341 bp in length encoded 446 amino acids. The deduced amino acid sequence demonstrated the cytochrome b5 domain in the N terminal and three conserved histidine boxes in the sequence. Then the gene was inserted into the expression vector pPIC3.5K and transformed into Pichia pastoris GS 115. Transformants containing multi-copy Δ5 desaturase genes were screened by Geneticin （G418）. When dihomo-γ-1inolenic acid was provided as the exogenous substrate to the culture induced by methanol, the dehydrogenation of the substrate was catalyzed by Δ5 desaturase to produce arachidonic acid reaching 2.85% in the total fatty acids. This result indicated that the cloned gene encoded functional Δ5 desaturase in P. pastoris GS 115.

关 键 词：高山被孢霉 Δ5去饱和酶基因 毕赤酵母 花生四烯酸 

分 类 号：Q78[生物学—分子生物学]