缺刻缘绿藻磷脂酶A2(PLA2)的基因特征与功能

为进一步鉴定磷脂酶A2(PLA2)在缺刻缘绿藻(Myrmecia incisa)油脂合成过程中的具体功能,研究利用RACE技术从缺刻缘绿藻H4301中克隆到pla2基因(Mipla2)的cDNA序列全长,共1082 bp。该全长包含180 bp的5′-非翻译区(UTR), 464 bp的3′-UTR和438 bp的开放阅读框(ORF),编码1个由145个氨基酸组成的蛋白。其基因全长为1594 bp,含有3个内含子和4个外显子。多序列比对结果显示, MiPLA2具有保守的Ca²⁺结合基序和催化活性基序。系统演化分析结果显示MiPLA2属于植物s PLA2-XIA家族。利用同源重组技术构建了pET32aMipla2原核表达载体,经过诱导和纯化,得到了分子量大小为31.36 kD的重组MiPLA2蛋白。体外酶促反应的产物经薄层层析分析显示,重组表达的MiPLA2具有催化磷脂酰胆碱(PC)水解成溶血磷脂酰胆碱(LPC)的功能。研究结果有助于解析花生四烯酸被优先用于三酰甘油(TAG)合成的途径与机理,为通过pla2基因的遗传改造提高该藻油脂产率的应用研究奠定了基础。

CHARACTERIZATION AND FUNCTIONAL IDENTIFICATION OF A PHOSPHOLIPASE A2 GENE FROM THE ARACHIDONIC ACID-RICH GREEN MICROALGA MYRMECIA INCISA REISIGL

Myrmecia incisa is a kind of green alga, which was rich in arachidonic acid (ARA). On the basis of the available reports, it was speculated that phospholipase A2 (PLA2) might play an important role in the lipid synthesis of M. incisa. In order to identify the function of PLA2, the full- length cDNA sequence of PLA2 gene (MiPLA2) of this alga was cloned by RACE technology. The full- length cDNA sequence of MiPLA2 was 1082 bp in length, and consisted of a 5′-untranslated region (UTR) of 180 bp, a 3′-UTR of 464 bp, and an open reading frame (ORF) of 438 bp, which encoded a protein of 145 amino acids. The full-length DNA sequence of MiPLA2 gene was 1594 bp, which contained 3 introns and 4 exons. Multi-sequence alignments of the secreted PLA2 (sPLA2) proteins from several different species of plants showed that MiPLA2 harbored a conserved Ca2+ binding motif and a catalytically active domain. Phylogenetic analysis illustrated that MiPLA2 belongs to the plant sPLA2-XIA family. Then, the pET32a-Mipla2 prokaryotic expression vector was constructed. After induction and purification, a recombinant MiPLA2 protein with a molecular weight size of 31.36 kD was obtained. Thin-layer chromatography (TLC) analysis of the in vitro enzymatic reaction products showed that the recombinant MiPLA2 could catalyze the hydrolysis of phosphatidylcholine (PC) into hemolytic phosphatidylcholine (LPC). The results might help to reveal the pathway and mechanism of ArA being preferentially used for triacylglycerol (TAG) synthesis, and lay a foundation for improving the oil production in this alga by the genetic modification of pla2 gene.