棉花磷脂酶C基因的克隆及参与油脂代谢的功能分析

Cloning of a Phospholipase C Gene fromGossypium hirsutum and Its Functional Analysis of Participating in Lipid Metabolism

以陆地棉胚珠和纤维为实验材料,利用RT-PCR技术克隆得到磷脂酶C(Phospholipase C,PLC)基因(GhPLC,GenBank登录号:KR154219),将棉花磷脂酶C基因转化拟南芥,分析其在油脂代谢过程中的重要作用。测序鉴定显示,GhPLC基因的全长开放读码框为1 524bp,编码包含508个氨基酸的蛋白质,理论分子量约为55kD。序列比对分析显示,GhPLC属于典型的碱性磷酸酶超家族的磷脂酶。利用pET32a-GhPLC原核表达获得分子量约为55kD左右的重组蛋白GhPLC;酶活力分析显示,重组GhPLC蛋白具有较高的将卵磷脂(PC)催化为二酰甘油(DAG)的酶活力。半定量RT-PCR结果表明,GhPLC基因参与棉花种子和纤维发育过程。构建植物过量表达载体35S∷GhPLC并转化哥伦比亚野生型拟南芥,转基因拟南芥中GhPLC基因的表达和PLC酶活力显著提高,且转基因拟南芥种子的油脂含量提高了5.3%。

A Gossypium hirsutum phospholipase C （GhPLC,GenBank Accession Number：KR154219） gene was cloned using RT-PCR method from developing ovule and fiber tissues,and the function of lipid metabolism was analyzed through transforming GhPLC into Arabidopsis.Sequencing identification showed that the full open reading frame of GhPLC was 1 524 bp containing a protein of 508 amino acids with a theoretical molecular weight of 55 kD.Sequence alignment analysis displayed that GhPLC gene belongs to the classical alkaline phosphatase superfamily.The recombinant proteins of GhPLC was obtained with molecular weight of 55 kD by prokaryotic expression of pET32a-GhPLC.Enzyme activity determination showed that recombinant GhPLC proteins has high activity of catalyzing phosphatidylcholine （PC） to diacylglycerol （DAG）.Semi-quantitative PCR analysis indicated that GhPLC gene participates in cotton seed and fiber development.Plant overexpression vector 35S∷GhPLC was constructed and transformed into Columbia wild-type Arabidopsis.Transgenic GhPLC Arabidopsis showed significant promotion both in transcription and enzyme levels,and the oil content of transgenic Arabidopsis seeds increased with 5.3% higher than wide-type.