Cloning And Function Study Of △6-Fatty Acid Desaturase Gene From Mucor.pusillus

Polyunsaturated fatty acids (PUFAs) plays important roles as some structural components of membrane lipids,maintain normal metabolism and the signal conduction in eukaryotic cells. Theγ-linolenic acid is one kind of essential unsaturated fatty acid , Long-chain polyunsaturated fatty acids (LCPUFAs) could be synthesized from GLA in body, these LCPUFAs is the important precursor of prostaglandin,prostacyclin and leukotriene and so on which have the intense biological activity .The research confirmed that some sicknesses such as high blood fats level,diabetes,arteriosclerosis,some kind of cancer,early aging are all related with the lacking these essential fatty acid .Takes in the right amount of essential fatty acid is important for adjusting the body's cholesterol metabolism,enforcing immunity function and improving the fatty acid metabolism.Mucor is a kind of fungus may produce the Long-chain polyunsaturated fatty acids, The research confirmed that Mucor could produceγ-linolenic acid. In This study we used a strain of GLA produceing Mucor, through PCR method we got aΔ6-fatty acid desaturase gene of Mucor. pusillus. And using the yeast expresses system pYES2.0 to confirm that this gene can cause the yeast to synthesis GLA.The main work and the result are as follows:1. Cloning and sequence analysis ofΔ6-Fatty acid Desaturase genePrimers were designed through comparative squences of other Mucor such as Mucor rouxii, ect. The PCR amplification of MpD6D using DNA and cDNA as template produced respectively a band of about 1.6 kb which was proved to be 1569 bp by sequencing, sequence anathyze approved that there was no integron in MpD6D.NCBI blastx (Translated query vs. protein database) analyse showed this fragment has 85% identities of nucleic acid sequence and 95% consistency of amino acid sequence toΔ6-desaturase from Mucor rouxii and has broad homology toΔ6-desaturases from other fungi and plants. These data suggested that we successfully cloned a novelΔ6-desaturase from Mucor.pusillus.2. Bioinformatical characteristics ofΔ6-desaturase geneThe bioinformatical characteristics of MpD6D were studied using online server or bio-soft,the protein homologous analysis, evolution analysis, motif analysis and quadruple structure predicting were carry out. Bioinformatics analysis indicated that the putative MpD6D protein is a typical membrane-bound desaturase with three conserved histidine-rich motifs,a cytochrome b₅ -like domain in the N-terminus. In addition to conserved histidine-richmotifs I, II and III corresponding to the sequencesHXXXH, HXXHH and QXXHH, respectively, we found an unusual histidine-rich motif (HKHHSH, positions 141-146) downstream of the cytochrome b₅ domain, which has not been reported previously in otherΔ6-desaturase genes. When expressed in Saccharomyces cerevisiae strain INVScl,it exhibited aΔ6-fatty acid desaturase activity which lead to the accumulation ofγ-linolenic acid. This functional MpD6D gene cloned here belongs to a novelΔ6-fatty acid desaturase of Mucor.pusillus. This was the first report on the characterization and gene cloning ofΔ6-fatty acid desaturase from Mucor.pusillus, which broaden a way for genetic engineering ofγ-linolenic acid and deep research onΔ6-fatty acid desaturase gene family,and lay certain foundation for further uses this gene to improve the crops quality.3. Recombinant vectorThe ORF fragment was cut off from recombinant pMD18-T using BamHI,KpnI digestion, recovered and was ligated to yeast expression vector pYES2.0 (Invitrogen, USA) digested with the BamHI,KpnI to generate a recombinant vector pYMpD6D.4. Yeast expressionThe recombinant plasmid pYMpD6D was transformed into S. cerevisiae INVScl using lithium acetate method for expression. After 8h cultivation, the yeast cells were induced with 2% galactose, supplemented with LA as substratethen the fats component yeast cells was anrysized by GC analysis .The result showed that a novel fatty acid methyl ester peak corresponding to GLA methyl ester standard was detected in the cultivated yeast cell transformed with pYMpD6D, which was absent in the yeast containing only vector pYES2.0. The percentage of this new fatty acid was 6.53% of total fatty acids. These results showed that pYMpD6D encoded aΔ6-fatty acid desaturase and the enzyme could convert the incorporated LA. to GLA specifically.