Cloning and Identification of △~6-fatty Acid Desaturase Gene of and Its 5′ Flanking Region from Mucor Sp.EIM-10

Abstract: γ-linolenic acid(GLA) is an important polyunsaturated fatty acid,which plays lots of physiological functions.EIM-10 accumulating significant amounts of GLA was isolated by using sudanblack staining.The oil was extracted through Acid-hydrolysis Method.The fatty acids were analyzed by gas chromatography mass spectrometry(GC/MS) after methyl esterification,γ-linolenic acid(GLA) was about 27.68%of the total fatty acids. The strain was further identified on the molecular level,the 18S rRNA gene of EIM-10 was cloned and sequenced.The phylogenetic analysis based on its 18S rRNA sequence showed it to be Mucor sp.The△~6-desaturase gene from Mucor sp.EIM-10(MCD6) was successfully cloned by RT-PCR.The PCR product was ligated into the yeast expression vector pYES2.0 to generate a recombinant plasmid pYMCD6,which was subsequently transformed into Saccharomyces cerevisiae strain INVScl.The recombinant yeast cells were selected on uracil-deficient medium.On appropriate medium and temperature,linoleic acid(LA) was provided as a substrate to yeast cultures.The GC/MS analysis on the profile of the total fatty acid demonstrated that the noval peak for product GLA appeared in the transgenetic yeast compared with the control yeast containing the original vector pYES2.0.The results indicated that the protein encoded by MCD6 could specifically catalyze LA into GLA under the induction of galactose.To understand the molecular mechanism of transcription regulation of△~6-desaturase gene,a 5'flanking region of MCD6 of 696 bp was cloned 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.The results indicated it was potential promoter region of MCD6.The MCD6 promoter sequence of 599 bp was then submitted to the GenBank database.Based on the shuttle vector pYES 2.0,we contructed the shuttle vector pYGFP with the GFP reporter gene,which could identify the promoter function of eukaryotic gene sequence quickly,simply and conviently.So the shuttle vector pYGFP would have a good appilication,which could be amplified in E.coli and be expressed in S.cerevisiae.Then the shuttle vector pYGFP was used to identify the promoter function of the 5' flanking region of MCD6,the 5'flanking region of MCD6 was subcloned into the shuttle vector pYGFP to generate a plasmid designated pYD6GFP.The recombinant vector pYD6GFP was transformed into S.cerevisiae with the same method ahead.Positive transformants were selected and cultured in SC-Ura culture.Afer 24-30 hours,we observed the cells under a fluorescence microscope with the proper exciting light and the positive tranformants gave off bright green fluorescence under the fluorescence microscope.The result showed that the 5'flanking region of MCD6 had the promoter function and could promote the expression of GFP gene in S.cerevisiae.Then,the recombinant plasmid pYD6MCD6 was constructed where MCD6 was cloned into downstream of the 5'flanking region of it,the recombinant plasmid pYD6MCD6 was then transformed into S.cerevisiae.After screening and culturing with the same method,fatty acids of positive yeast transformants were extracted and methyl-esterified as described previously.The fatty acid methyl esters were analyzed by GC/MS.The results showed that the promoter region of MCD6 also could drive the expression of MCD6 in S.cerevisiae…
Key words: γ-linolenic acid; GLA; △~6-fatty acid desaturase; clone; expression; promoter

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