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
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
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DOI：
10.3724/sp.j.1231.2013.38654
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发表时间：
2013
期刊：
影响因子：
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通讯作者：
周志刚
中科院分区：
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作者：
房逢立;吴 洪;周志刚
为了2.3.1.20研究桃金娘（Myrmagnifisa）的三酰甘油（TAG）合成途径，我们对桃金娘的转录组进行了焦磷酸测序，得到了一个DGAT 2的cDNA全长序列，该序列全长1 997 bp，包含一个44 bp的5′非翻译区（UTR），该基因3′-UTR为897 bp，具有典型的poly A尾，开放阅读框（ORF）为1056 bp，编码351个氨基酸，分子量为39.43 ku，pI为9.46。与DGAT 1和DGAT 3家族的DNA序列相比，该基因具有DGAT 2家族的特征性保守序列HPHG，因此将该基因命名为MiDGAT 2。结果表明，MiDGAT 2的编码区被6个内含子打断，所有的剪接位点都符合GT-AG规则。通过RT-PCR扩增其开放阅读框，亚克隆到穿梭载体pYES 2中，构建重组载体pY-MiDGAT 2重组质粒通过电穿孔转化到酿酒酵母TAG缺陷突变体H1246中进行表达，测序证实目的基因已整合到酵母基因组中，转化子中pY-将该酵母菌在含有半乳糖诱导剂的SC培养基中培养，通过薄层色谱分析表明，该酵母菌的TAG-1、TAG-2、TAG-3、TAG-4、TAG-5、TAG-6、TAG-7、TAG-8、TAG-10、TAG-11、TAG-用MiDGAT 2转化的缺陷突变体H1246可以恢复合成TAG的能力，表明MiDGAT 2编码参与TAG生物合成的DGAT酶。当用荧光染料Bodipy染色酵母细胞时，发现在用MiDGAT 2转化的TAG缺陷型突变体H1246中存在脂滴，尽管脂滴的直径小于野生型。
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.