花生及其野生种质溶血磷脂酸酰基转移酶基因(LPAAT)的克隆及序列分析

Cloning and Sequence Analysis of a Lysophosphatidic Acid Acyltransferase Gene(LPAAT) in Arachis hypogaea L. and Wild Diploid Species

溶血磷脂酸酰基转移酶(LPAAT)是植物种子油脂合成的关键酶。本研究从花生(Arachis hypogaea L.)栽培品种花育32号和4个花生区组二倍体野生种中克隆得到编码LPAAT的基因序列。从花育32号获得的两条cDNA序列rlpaat-1和rlpaat-2开放阅读框均为1 131 bp,编码376个氨基酸,二者存在11个SNP差异位点,编码的氨基酸序列LPAAT-1和LPAAT-2有1个氨基酸残基的差异。LPAAT蛋白具有典型的酰基转移酶功能结构域以及4个保守氨基酸基序。之后从花育32号得到两条DNA序列,glpaat-1和glpaat-2长度分别为3 729 bp和3 736 bp,均由12个外显子和11个内含子组成,二者共存在37处碱基差异,其中34处为SNP位点。4个花生区组二倍体野生种各获得一条LPAAT序列,A.correntina,A.duranensis,A.batizocoi和A.ipaensis LPAAT的序列长度分别为3 757 bp、3 757 bp、3 742 bp和3 756 bp。核苷酸序列多态性和系统进化树分析表明,栽培品种LPAAT的两条序列glpaat-2与glpaat-1分别来自A、B染色体组。

Lysophosphatidic acid acyltransferase （LPAAT） is a pivotal enzyme responsible for the acylation of ly- sophosphatidic acid （LPA） into phosphatidic acid （PA）. In this study, we obtained the sequences of its encoding gene LPAAT from one cultivated variety and four wild diploid species. Two cDNA sequences rlpaat-1 and rlpaat-2 were isolated from Huayu32 and they both had an ORF （open reading frame） of 1 131 bp which encodes a putative protein of 376 amino acid residues. There were 11 different SNP （single nucleotide polymorphism） sites within rlpaat-1 and rlpaat-2, leading to one amino acid difference in the corresponding amino acid sequences LPAAT-1 and LPAAT-2. LPAAT protein comprised a conserved acyltransferase domain and four conserved motifs that existed in the whole acyltransferase family. Then DNA sequences were cloned from Huayu32 and four wild species. The length ofglpaat-1 and glpaat-2 obtained from Huayu32 were 3 729 bp and 3 736 bp, respec- tively. Sequence alignment revealed that between glpaat-1 and glpaat-2, there were 37 different sites in total, of which 34 were SNP sites. But they both had 12 exons and 11 introns. One sequence was obtained from each wild species, for A. correntina, A. duranensis, A. batizocoi, A. ipaensis, the sequence length ofLPAA T was 3 757 bp, 3 757 bp, 3 742 bp and 3 756 bp, respectively. Nucleotide polymorphism analysis and phylogenetic analysis indi- cated that glpaat-1 and glpaot-2 came from different genomes.