毛霉Δ6脂肪酸脱饱和酶基因启动子的克隆和功能鉴定-微生物学专业毕业论文.docxVIP

Abstract Abstract 脚YM1 ㈣8㈣0M6舢8舢9棚3 Abstract 7-1inolenic acid(GLA)is important polyunsaturated fatty acid，which plays lots of physiological functions．The A6-desaturase is the rate-limiting enzyme of GLA biosynthesis pathway． To understand the molecular mechanism of transcription regulation of A6一desaturase gene，a 5’ flanking region of MCD6 about 1 300 bp was specifically amplified by LA-PCR,then the sequence was analyzed by several soft：wares，online promoter prediction webs and aligned in several transcription factor databases and promoter databases to identify putative cisregulatory promoter elements such as TATA box，GC box，CAAT box like elements in the identical positions common to the eukaryote promoter sequence regions． According to the plasmid of PYESPMCD6 and PYGFP,the plasmid of PYMCD6 was constructed．The amplified product of promotor region was ligated into the PYMCD6 vector which contained the A6一fatty acid desaturase gene to construct the expression plasmid PYMD6PMCD6．The recombined plasmid was transferred into Saccharomyces cerev括iae strain INVScl．The GC／MS analysis the profile of the total fatty acid demonstrated that the novel peak for product GLA appeared in the transgenetic yeast compared with the control yeast containing the original vector PYMCD6． To understand the molecular mechanism of transcription regulation of A6一desaturase gene，The amplified sub-cloned products of the promtor region were ligated into the vector which contained the A 6·desaturase gene(MCD6)as the reporter gene to construct the expression plasmids．The recombined plasmid was transferred into Saccharomyces cerevisiae strain INVSc 1．The GC／MS analysis the profile of the total fatty acid showed that all different length fragments of A6- desaturase gene promotor except the··1 2 1 bp fragment could drived the expression of A6·-desaturase gene which converted LA to GLA．The substrate conversion rate decreased with the length of promotor changed from 1 29 1 bp to 434 bp．Howerer,the sta