缺刻缘绿藻二酰甘油酰基转移酶2（DGAT2）的基因特性与功能鉴定

Abstract

Acyl-CoA:diacylglycerol acyltransferase(DGAT;EC 2.3.1.20)is regarded as a key enzyme in triacylglycerol(TAG)biosynthesis of microalgae and other plants.In order to understand the biosynthesis of TAG in Myrmecia incisa,we found a putative full-length DGAT2 cDNA sequence in the homology search of a pyrosequencing transcriptome of this microalga.The full-length cDNA sequence was composed of 1 997 bp.It comprised a 44-bp 5′-untranslated region(UTR),a 897-bp 3′-UTR with a typical poly A tail,and a 1 056 bp open reading frame(ORF)encoding a 351-amino-acid protein with a putative molecular weight of 39.43 ku and pI at 9.46.Neighbor-Joining(NJ)phylogenetic tree inferred from the putative proteins of DGAT genes indicated that this gene belongs to DGAT2 gene family,significantly different from DGAT1 and DGAT3 families.Multiple sequence alignment of amino acids indicated that a conserved and characteristic sequence HPHG of the DGAT2's was present in this gene.Therefore,this gene was designated as MiDGAT2.Compared with the DNA sequence of MiDGAT2,it was found that its coding region was interrupted by 6 introns with all splicing sites well matching the GT-AG rule.In order to investigate the function of MiDGAT2,its open reading frame was amplified by RT-PCR and sub-cloned into the shuttle vector pYES2 to generate the recombinant vector pY-MiDGAT2.This recombinant plasmid was transformed into a TAG-defective mutant H1246 of Saccharomyces cerevisiae for expression by electroporation.The target gene integrated in the yeast genome was confirmed by sequencing and a transformant with pY-MiDGAT2 was screened out.This yeast transformant was cultured in SC medium with galactose as an inducer.Thin layer chromatogram(TLC)analysis of yeast lipids showed that the TAG-defective mutant H1246 transformed with MiDGAT2 could restore the ability to synthesize TAG,indicating that MiDGAT2 encodes a DGAT enzyme involved in the biosynthesis of TAG.When the yeast cells were stained with fluorescent dye Bodipy,it was found that lipid droplets were present in the TAG-defective mutant H1246 transformed with MiDGAT2,although the diameter of lipid droplets was smaller than that of wild type.