Identification and characterization of three genes encoding acyl-CoA: diacylglycerol acyltransferase (DGAT) from the microalga Myrmecia incisa Reisigl
Identification and characterization of three genes encoding acyl-CoA: diacylglycerol acyltransferase (DGAT) from the microalga Myrmecia incisa Reisigl
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编码酰基辅酶 A 的三个基因的鉴定和表征：来自微藻 Myrmecia incisa Reisigl 的二酰基甘油酰基转移酶 (DGAT)
DOI：
10.1016/j.algal.2015.09.007
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发表时间：
2015-11-01
影响因子：
5.1
通讯作者：
Zhou, Zhi-Gang
中科院分区：
文献类型：
--
作者：
Chen, Chun-Xiu;Sun, Zheng;Zhou, Zhi-Gang
酰基辅酶A：二酰基甘油酰基转移酶（DGAT）是真核生物中负责三酰基甘油（TAG）合成的关键酶。本工作报道了一种有希望的花生四烯酸（ArA）生产候选菌株--绿色桃金娘（Myrmartinincisa）的DGAT基因。根据转录组数据库的同源性搜索结果，我们克隆了三个编码DGAT 1和DGAT 2的cDNA。MiDGAT 1、MiDGAT 2A和MiDGAT 2B的开放阅读框（ORF）分别为2238 bp、1056 bp和1068 bp，编码由745、351和355个氨基酸组成的蛋白质。通过比较它们相应的cDNA和DNA序列，发现它们分别被14、6和6个内含子分开。多序列比对结果表明，MiDGAT 1含有一个普列克底物蛋白同源结构域（PH），而MiDGAT 2含有高度保守的DGAT 2家族特征性基序HPHG。为了确定其功能，在TAG代谢受损的酿酒酵母突变株中异源表达它们。薄层层析和BODIPY染色结果表明，MiDGAT 1和MiDGAT 2都能够恢复TAG合成和脂质体形成。GC-MS分析表明，棕榈酸和硬脂酸是酵母细胞TAG的主要成分，其比例在野生型和转化酵母之间没有显著差异。定量RT-PCR结果表明，氮饥饿对MiDGAT 2A的转录水平有调节作用，这与M.切口(C)2015作者由Elsevier B出版。诉
Acyl-CoA: diacylglycerol acyltransferase (DGAT) is a key enzyme responsible for triacylglycerol (TAG) synthesis in eukaryotic organisms. The present work reported DGAT genes in the green alga Myrmecia incisa, a promising candidate for arachidonic acid (ArA) production. According to the results of homology search against a transcriptome database, we cloned three cDNAs encoding putative DGAT1 and DGAT2. The 2238-bp, 1056-bp and 1068-bp of open reading frame (ORF) of these three cDNAs, designated as MiDGAT1, MiDGAT2A and MiDGAT2B, were predicted to encode proteins composed of 745, 351 and 355 amino acids. They were separated by 14, 6 and 6 introns, respectively, as revealed by comparing their corresponding cDNA and DNA sequences. Multiple sequence alignment of amino acids indicated that MiDGAT1 had a pleckstrin homology (PH) domain, whilst MiDGAT2s contained a highly conserved HPHG, a characteristic motif of DGAT2 family. To determine the function, they were expressed heterologously in a Saccharomyces cerevisiae mutant strain with impaired TAG metabolism. Results of thin-layer chromatography and BODIPY staining indicated that both MiDGAT1 and MiDGAT2s were able to restore TAG synthesis and lipid body formation. GC-MS analysis indicated that palmitic acid and stearic acid were the major components of TAGs in yeast cells, and their ratio between wild type and the transformed yeasts was not significantly different. Quantitative RT-PCR results showed that the transcript level of MiDGAT2A was regulated by nitrogen starvation, which was consistent with TAG accumulation in M. incisa. (C) 2015 The Authors. Published by Elsevier B. V.