Cloning And Functional Analysis Of Genes Encoding Key Enzymes In Sphingolipids Biosynthesis Pathway In Emiliania Huxleyi Virus

Sphingolipids are essential constituents of eukaryotic cells.Besides playing structural roles in cel ular membranes,some metabolites have drawn attention as bioactive signaling molecules.As natural ingredients in food,sphingolipids have been considered as "functional nutrient ingredients".Emiliania huxleyi(coccolithophores)is a marine eukaryotic microalgae.It can produce abundant secondary metabolism active substances.Recently,an unusual sphingolip id from virus infected E.huxleyi was isolated and analyzed,implicating a novel structure with highly bioactivity,which will have important scientific significance and broad application prospects in the field of food nutrition research.The E.huxleyi virus(EhV-86)genome sequence has identified and several genes apparently involved in sphingolipid metabolism,such as serine palmitoyltransferase(SPT),Longevit yassurance family protein(LAG1),fatty acid desaturase(FAD),lipid phosphate phosphatase(LPP),sterol desaturase(SD),Glycosyltransferase(GT)),transmembrane fatty acid elongationprote i n(TFAEP).The EhV-86-encoded genes contain only a subset of the activities required to generate the novel sphingolipid,implying that its synthesis is the result of coordinated interactions between algal-and viral-encoded biosynthetic enzymes.So far,only the SPT biochemical characterist ics and functions has been studied preliminarily.The exact locations and functions of several other enzymes in the sphingolipid metabolic pathway remained unknown.In this paper,two kinds of the key enzymes,FAD and SD from EhV99B1 were selected as the study subjects.The function of SD and FAD were analyzed by using genetic engineer i ng technique..Based on this research,it is expected to provide scientific basis for the use of sphingolipids-rich foods or supplements to promote health and prevent the nutrient-rela ted diseases.The main results are as follows:(1)SD and FAD genes were isolated by PCR.Bioinformatics analysis results showed: the SD gene had a open reading frame of 987 bp containing a deduced amino acid sequence of 327 residues with a calculated molecular mas of 37.83 kDa and its theoretical pI was 8.31.Subcellular localization suggested that SD may be a mitochondrial product protein.SD had six transmemb ra ne domains without signal peptide and the helical was dominated in the secondary.FAD gene had a open reading frame of 963 bp containing a deduced amino acid sequence of 320 residues with a calculated molecular mas of 37.67 kDa and its theoretical pI was 7.26.FAD had four transmembrane domains without signal peptide and the helical was dominated in the secondary.The phylogenetic analysis indicated that the SD and FAD from EhV was closer to E.huxleyi and Chrysochromulina.(2)The PCR products were digested and subcloned into pYES2.0/CT to generate two plasmid designated pYES2/CT-SD and pYES2/CT-FAD respectively.Sacharomyces cerevisiae was transformed with pYES2/CT-SD pYES2/CT-FAD and pYES2/CT,using the lithium acetate method.Transformants were selected by plating on complex synthetic minimal mediun agar lacking uracil(SC-Ura-).The pulative SD and FAD gen were heterologously expressed in yeast S.cerevisae strain INVSc1 and the recombinant proteins were detected by SDS-PAGE.The proteins were 37.8 kDa(SD)and 37.6 kDa(FAD)respectively.Then the total lipids extracted from the cells were analyzed by using thin layer chromatography(TLC).There was a significant differe nce of the total lipids between the yeast cells conaining the recombinant vectors and the wild yeast cells.It indicated that SD and FAD genes could make lipids accumulate in yeast cells.(3)The FCP-P,FCP-T,Bar and GFP genes were cloned by using genetic engineering technology from commercial available plasmids respectively.Eukaryotic expression vectors psp73-FAP-Bar-FAT1-FBP-FAT2 and psp73-FBP-GFP-FAT2 were constructed by using different endonucleasea.The two vectors were co-transformed into E.huxleyi cells by PEG-mediated transfer.The green fluorescent labbled cells were observed by using fluorescence microscopy and the positive cells were screened by using glufosinate.Real-time quantitative PCR and western blotting were used to confirm the successful transformation of the constructed vector into algal cells.