Cloning And Function Identification Of The Promoter Of Δ6-Fatty Acid Desaturase Gene From Mucor Sp.

y-linolenic acid (GLA) is an important polyunsaturated fatty acid, which plays lots of physiological functions. TheΔ6-desaturase is the rate-limiting enzyme of GLA biosynthesis pathway. To understand the molecular mechanism of transcription regulation ofΔ6-desaturase gene, a 5' flanking region of MCD6 about 1300 bp was specifically amplified by LA-PCR,.then the sequence was analyzed by several softwares,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 theΔ6-fatty acid desaturase gene to construct the expression plasmid PYMD6PMCD6. The recombined plasmid was transferred into Saccharomyces cerevisiae strain INVSc1. The GC/MS analysis on 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 ofΔ6-desaturase gene, The amplified sub-cloned products of the promtor region were ligated into the vector which contained theΔ6-desaturase gene (MCD6) as the reporter gene to construct the expression plasmids. The recombined plasmid was transferred into Saccharomyces cerevisiae strain INVScl.The GC/MS analysis on the profile of the total fatty acid showed that all different length fragments ofΔ6-desaturase gene promotor except the-121 bp fragment could drived the expression ofΔ6-desaturase gene which converted LA to GLA. The substrate conversion rate decreased with the length of promotor changed from 1291 bp to 434 bp. Howerer, the stability of substrate conversion rate decreased sharply under the condition of lacking the-919 bp～-784 bp fragment of promtor, which showed that the the-919 bp～-784 bp fragment may have the function of enhancer.To confirm and research the enhancer function of-919 bp～-784 bp fragment, the fusion fragment containing the-919 bp～-784 bp and-434 bp～-1 bp fragments was amplificated using Overlap PCR. Then the fusion fragment bonded to PYMCD6 vector to construct the expression plasmid PYOL94MCD6. The recombined plasmid was transferred into Saccharomyces cerevisiae strain INVSc1. The GC/MS analysis on the profile of the total fatty acid showed that both of the fusion fragment and the-434 bp～-1 bp fragment ofΔ6-desaturase gene promotor could drived the expression ofΔ6-desaturase gene which converted LA to GLA. However,the substrate conversion rate increased sharply with the fusion fragment rather than the-434 bp～-1 bp fragment.