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.