Cloning and Identification of △5 Fatty Acid Desaturase Gene and Its 5′ Flanking Region from Thraustochytrium

Abstract: A cDNA with homology to fatty acid desaturases was selected fromThraustochytrium sp.FJN-10 (GenBank Accession Number: EU643618) , which containsan N-terminal cytochrome b5-like domain as well as three histidine-rich domains that is acommon feature of primary structure of fatty acid desaturase, the third histidine boxcontains a histidine to glutamine substitution(HXXHH–QXXHH). The coloned ORF was1320bp encodes a protein with 439 amino acids, its molecular weight is 49.82 KDa, andthe pI is 8.71. Homology analysis suggest the sequence was 99% identities with theΔ5-desaturase of Thraustochytrium sp. ATCC21685 and 49%, 47% with theΔ5-desaturase of Pavlova salina, Perkinsus marinus respectively, the sequence alsoshare 42%, 41% identities with theΔ6-desaturase of Mantoniella squamata andOstreococcus tauri respectively. Its desaturase activity was confirmed by expression of itunder the inducible promoter of galactose in Saccharamyces Cerevisiae in the present ofDGLA as substrate, total lipids were extrated from them, a new fatty acid was detected inthe lipid fraction by GC-MS (gas chromatography-mass spectrum), the results suggestedthe recombinant cell converted the DGLA to AA, with a conversion efficiency of 56.40%.Therefore, the cDNA cloned from Thraustochytrium sp.FJN-10 expressed in S. cerevisiaecan introduce aΔ5 double bond into DGLA, converted DGLA to AA, this result indicateditsΔ5-desaturase activity in S. cerevisiae.The 5' flanking region ofΔ5-desaturase gene was cloned by LA-PCR and sequenced (GenBank Accession Number: EU918393), then it was analyzed by many on-line softs which all suggested the region contains many significant characteristics of promoter. Characterization and comparison of this sequence with the 5' flanking region ofΔ5-desaturase gene from Human, Zebrafish, Rhesus, C.elegan, Rattus norvegicus, phylogenetic tree was constructed by Neighbor-Joining method , the result showed that Thraustochytrium sp.FJN-10 share the smallest evolution distance with Human and Rhesus which implied Thraustochytrium sp.FJN-10 may share the same regulation mechanism of the expression ofΔ5-desaturase gene with higher vertebrates, this conclusion was supported by FootPrinter analysis again. In order to verified the promoter function of the 5' flanking region ofΔ5-desaturase gene, we have constructed a promoter function identification vector pYGFP, ligated this sequence with pYGFP vector which located at the upstream of GFP to constructed recombinant plasmid pYD5GFP, then pYD5GFP was transformed into Saccharamyces Cerevisiae. The GFP(green fluorescent protein) gene was expressed in Saccharamyces Cerevisiae under the regulation of this sequence, fluorescence was assayed by microscope. The result suggested that the recombinant cells yielded a green fluorescent when they were excited by the corresponding wavelength light which indicated its full function of promoter…
Key words: Δ5-desaturase; Promoter; Cloning and expression; Thraustochytrium

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