高山被孢霉Δ~6-脂肪酸脱氢酶基因的克隆、结构分析及其功能的研究  

Studies on the Cloning, Structure and Function of the Δ~6-Fatty Acid Desaturase Genes from Mortierella aplina

作　　者：李明春[1] 刘莉[1] 胡国武[1] 邢来君[1] 

机构地区：[1]南开大学微生物系,天津300071

出　　处：《微生物学报》2003年第2期220-227,共8页Acta Microbiologica Sinica

基　　金：国家自然科学基金 (3 9870 0 2 0 );教育部高等学校骨干教师资助计划项目~~

摘　　要：从高山被孢霉ATCC1 62 66总DNA中扩增出大小为 1 374bp和 1 947bp的两条特异片段 ,序列分析表明后者在细胞色素b5和组氨酸Ⅰ之间含有一大小为 5 73bp的内含子和两条分别为 1 97bp和 82 8bp的外显子。推导的氨基酸二级结构分析表明 ,该基因有两个长的跨膜疏水区和 3个组氨酸保守区。分别根据D6D内含子及组氨酸Ⅱ区、Ⅲ区的序列设计引物 ,制备不同的探针与高山被孢霉的基因组杂交 ,证明在其基因组中确实存在两个Δ6 脂肪酸脱氢酶基因 ,其中一个基因含有内含子。把不含有内含子的核基因MAGL6 1克隆到的酿酒酵母表达载体pYES2 0中 ,转化到酿酒酵母INVSc1中。对筛选得到的酵母工程菌株进行脂肪酸GC分析 ,检测到了γ 亚麻酸 ,说明克隆的D6D基因MAGL6 1能在酿酒酵母中进行功能性表达。The genomic genes were cloned from the fungal strain, Mortierella aplina ATCC16266,through PCR with primer pairs designed on information of conserved region of the Δ 6 fatty acid desaturase genes from the several different speices. Results show genomic DNA sequences in 1347bp and 1947bp respectively. Comparison of the genomic and cDNA sequences revealed that the D6D gene from genomic DNA had an 573bp intron, ranging from 196 to 769 nucleotides in length and locating of between the Cytb 5 and HisⅠregions. The exons are 196bp and 828bp. Furthermore, genomic southern blot analysis with the M.alpina Δ 6 fatty acid desaturase cDNA and intron fragments as the probes respectively confirmed that there are two D6D genes in M.aplina. The one gene doesn′t contain a intron named MAGL 1, another gene contains a intron named MAGL 2 Then, the gene MAGL 1 was subcloned into the yeast E.coli shuttle vector pYES2 0,thus expression recombinant plasmid containing M.alpina ATCC16266 D6D gene was constructed. The plasmid was introduced into Saccharomyces cerevisiae for expression by LiAc method. When linoleic acid was provided as an exogenous substrate to the yeast culture expressing Δ 6 fatty acid desaturase activity under appropriate media and temperature conditions, the recombinant S.cerecisiae had γ linolenic acid by GC detecting, not normally present in wild type yeast cells. This results indicated that the gene encoded functional Δ 6 fatty acid desaturase. It is first report that there are two D6D genes in M.aplina.

关 键 词：高山被孢霉 △^6-脂肪酸脱氢酶基因 基因组 内含子 功能 克隆 结构 

分 类 号：Q786[生物学—分子生物学]