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

Characterization and functional identification of an acyl-CoA:diacylglycerol acyltransferase 2(DGAT2)gene from the green microalga Myrmecia incisa

酰基-CoA—二酰甘油酰基转移酶(DGAT)是微藻等植物三酰甘油(TAG)合成过程中的关键酶。为了解析缺刻缘绿藻TAG合成代谢的途径,在该藻转录组测序数据库中,通过同源搜索,发现了1个编码DGAT2的cDNA全长序列。该序列长1 997 bp,其中5'-非翻译区(UTR)长44 bp,3'-UTR长897 bp,开放阅读框(ORF)长1 056 bp,编码1个含有351个氨基酸的蛋白质,预测的分子量为39.43 ku,等电点为9.46。基于缺刻缘绿藻和其他物种相应的DGAT基因编码蛋白序列所构建的Neighbor-Joining(NJ)系统进化树,结果表明该基因与DGAT2聚成一支,显著不同于DGAT1和DGAT3。氨基酸序列比对发现,该基因编码产物含有DGAT2所具有的HPHG这4个氨基酸所组成的高度保守特征序列。因此,将该基因命名为MiDGAT2。将它的cDNA与其DNA序列进行比较后发现,MiDGAT2含有6个内含子,其剪接位点均符合"GT-AG"规则。为进一步了解其功能,利用反转录PCR克隆了该基因的ORF序列,然后将其亚克隆到表达载体pYES2中,成功地构建了重组表达质粒pY-MiDGAT2。通过电穿孔法将该重组质粒转入酿酒酵母TAG合成缺陷株H1246中,经筛选与序列验证得到含有重组质粒pY-MiDGAT2的酵母转化株。酵母转化株在用SC培养基并加入半乳糖诱导表达培养后,其脂类的薄层色谱分析结果表明,所转的MiDGAT2基因能使酵母转化株恢复TAG合成的能力,从而证实了MiDGAT2基因具有DGAT的功能;利用荧光染料Bodipy对酵母细胞的染色结果显示,MiDGAT2基因能使酵母转化株的细胞重现油滴,尽管重建的油滴大小明显比野生型酵母的小。

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 pl 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 MiDGAI2, 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.