高山被孢霉Δ5脱饱和酶基因的分离与验证  

Isolation and Functional Identification of Δ5 Desaturase Gene from Mortierella alpina

作　　者：朱敏[1] 刘智[1] 余龙江[1] 朱路[1] 程华[1] 

机构地区：[1]华中科技大学生命科学与技术学院,武汉430074

出　　处：《Acta Genetica Sinica》2005年第9期986-992,共7页

摘　　要：花生四烯酸(arachidonicacid)是人体的必需脂肪酸,具有独特的生物活性。Δ5脱饱和酶是花生四烯酸生物合成途径上的关键酶,以二高-γ-亚麻酸为底物,催化其第5位碳脱氢形成花生四烯酸。从花生四烯酸高产菌株高山被孢霉M6(Mortierellaalpina)中通过RT-PCR分离了可能的编码Δ5脱饱和酶完整的cDNA,其大小为1366bp,编码446个氨基酸。推导出的蛋白质含有Δ5脂酰脱饱和酶中保守的特征性结构域,包括该蛋白质N端典型的细胞色素b5结构域,以及3个保守的组氨酸盒。为验证该蛋白质的功能,将该基因片段克隆到穿梭表达载体pPIC9K中,获得的重组载体pPIC9K-D5通过电转化法转化到巴斯德毕赤酵母GS115中,利用G418耐受度筛选出高拷贝数的酵母转化子,在加入外源底物二高-γ-亚麻酸时,利用甲醇诱导酵母转化子表达外源基因。酵母转化子的油脂的气相色谱图谱中出现了一个花生四烯酸的特征峰,进一步对该峰进行气质联谱(GC-MS)分析,证实该峰是花生四烯酸。研究结果表明,从高山被孢霉M6中分离出Δ5脱饱和酶基因。Arachidonic acid is an essential fatty acid in human nutrition and a biogenetic precursor of the biologically active prostaglandins and leukotrienes. △5 desaturase is a key enzyme in the biosynthetic pathway of arachidonic acid, which catalyze the △5 dehydrogenation of di-homo-γ-linolenic acid to form arachidonic acid. Complete cDNA encoding putative △5 fatty acyl desaturase was isolated from Mortierella alpina M6 via RT-PCR. The full length cDNA consisted of 1366 nucleotides encoding 446 amino acids. The deduced protein had conserved domains of known △5 fatty acyl desaturases including a cytochrome b5 domain in the N terminal and 3 conserved histidine box, To elucidate the function of the protein, the cDNA was subcloned into the expression vector pPIC9K. The resultant recombinant plasmid pPICgK-D5 was transformed to Pichia pastoris GS115 by electroporation. Transformants containing multi-copy △5 desaturase gene were screened by Geneticin resist- ance. The transformants were induced to express the inserted gene with methanol when di-homo-γ-linolenic acid was provided as an exogenous substrate. Analysis of the recombinant yeast lipids by gas chromatograph showed that a novel peak corresponding to the standard of arachidonic acid was detected. The novel peak was further characterized by GC-MS and identified as arachidonic acid. The results showed that the gene isolated from fungi Mortierella alpina M6 was a △5 desaturase gene.

关 键 词：花生四烯酸 高山被孢霉 △D5脱饱和酶基因 巴斯德毕赤酵母GS115 

分 类 号：Q789[生物学—分子生物学]