【作者】 刘建民；

【导师】 余龙江； 栗茂腾；

【作者基本信息】 华中科技大学 ， 生物化工， 2007， 硕士

【摘要】 γ-亚麻酸(γ-Linolenic acid,GLA)属于n-6系列多不饱和脂肪酸,是合成前列腺素E1和E2的前体,在维持人的正常生理功能中起着重要作用。高山被孢霉是花生四烯酸高产菌株,在其生物合成途径中含有γ-亚麻酸合成的关键酶-△6脂肪酸脱饱和酶。本文利用高山被孢霉W15为材料,克隆了△6脂肪酸脱饱和酶基因全长和△6脂肪酸脱饱和酶的cDNA序列,并对克隆的△6脂肪酸脱饱和酶基因进行了表达分析。△6脂肪酸脱饱和酶基因的全长为2202bp,该序列长度在高山被孢霉中是国内外最新报道,与已报道的序列同源性最高达99%;结构分析表明,该序列含有两个内含子,分别处于17-229bp和410-1024bp位置。以高山被孢霉W15 RNA为模板,通过RT-PCR扩增出可能的△6脂肪酸脱饱和酶基因,该基因全长为1374bp,推导的蛋白质序列编码457个氨基酸,与报道的高山被孢霉△6脂肪酸脱饱和酶基因的同源性达到95%,其编码的蛋白质N端具有典型的细胞色素b5结构域。将该可能的△6脂肪酸脱饱和酶基因连接到毕赤酵母表达载体pPIC9K上,通过电穿孔法转化到巴斯德毕赤酵母GS115中,1%的甲醇诱导转化子表达外源基因,通过对转化子油脂的气相色谱分析,与γ-亚麻酸标准品做对照,有γ-亚麻酸峰的出现,证明插入的基因为△6脱饱和基因酶。△6脂肪酸脱饱和酶基因的克隆与成功表达,为进一步筛选到高产γ-亚麻酸的菌种并应用于工业化生产,或转入油菜、大豆等油料作物中,以改变其脂肪酸组成,培育新型的作物品系提供前提和基础。更多还原

【Abstract】 Gamma linolenic acid, one of n-6 class polyunsaturated fatty acid, is precursor of prostaglandin E1 and prostaglandin E2 synthesis and plays an important role in keeping humans’normal physiological function. Mortierella alpina is highly productive strain of arachidonic acid.△6 fatty acid desaturase is a key enzyme of gamma linolenic acid synthesis in fatty acid biosynthetic pathway of Mortierella alpina. In this paper,△6 fatty acid desaturase gene full length sequence and△6 fatty acid desaturase gene cDNA have cloned from Mortierella alpina and expressed in Pichia pastoris GS115.△6 fatty acid desaturase gene consists of 2202 base pairs, which is the newest report in Mortierella alpina of home and abroad.. This sequence had the highest similarity with Mortierella alpina which had been reported with percent identities being 99. Structural analysis indicated this sequence contained two introns, locating in 17-229bp and 410-1024bp respectively. Total RNA was prepared from Mortierella alpina W15, possible cDNA fragment encoding△6 fatty acid desaturase gene was amplified by RT-PCR. The△6 fatty acid desaturase gene contained 1374 bp and codes 457 amino acids, sharing a 95% homology with other Mortierella alpina reported. The encoded protein consisted of typical cytochrome b5 domain in the N-terminal. The possible△6 fatty acid desaturase gene was cloned into plasmids pPIC9K and transformed into Pichia pastoris GS115 strain by the electroporation.. Induced by 1% methanol, exogenous gene was expressed and the converter oil was determined by GC. The results indicated that the sample hasγ-linolenic acid peak, comparing with positive control ofγ-linolenic acid standard, which certificated that the inserted gene was△6 fatty acid desaturase gene. The successful clone and expression of△6 fatty acid desaturase gene provide premise and foundation not only for sieving high-yieldγ-linolenic acid producing strains and further applying to industrialization, but also for transferring into oil grains, such as rape and soybean, in order to alter fatty acid composition and cultivate new type crops.更多还原