花生溶血磷脂酸酰基转移酶基因的克隆与表达分析

Cloning and Expression Analysis of Lysophosphatidic Acid Acyltransferase(LPAT) Encoding Gene in Peanut

溶血磷脂酰基转移酶(LPAT)是植物油脂合成途径的一个关键酶,在植物油脂品质改良和提高种子含油量方面具有重要的应用价值。本研究通过构建花生种子全长cDNA文库,结合大规模EST测序和功能注释,从花生中克隆了溶血磷脂酸酰基转移酶基因,命名为AhLPAT。该基因cDNA全长1629bp,对应的基因组序列5531bp,由11个外显子和10个内含子组成,内含子剪接方式符合GT-AG剪接规则。根据编码区预测AhLPAT编码一条387个氨基酸组成的多肽,预测分子量为43.2kD,等电点为9.42。AhLPAT蛋白含有一个典型的酰基转移酶保守功能结构域以及溶血磷脂酰基转移酶相似的保守区域。该蛋白的氨基酸序列与已报道的其他物种LPAT蛋白序列有较高的一致性。AhLPAT与旱金莲、油菜、海甘蓝、蓖麻和拟南芥的LPAT蛋白氨基酸相似性依次为90%、89%、89%、88%和87%。系统进化分析表明,AhLPAT与拟南芥AtLPAT2亲缘关系较近,且同属于内质网型LPAT蛋白。RT-PCR分析表明,AhLPAT基因在花生根、茎、叶、花、果针和种子中均有表达,在花生开花后50～60d,果针和种子中的表达量最高,且AhLPAT的表达量与花生种子含油量积累速率变化一致,二者显著相关(r=0.63,P<0.05)。推测AhLPAT基因在花生种子油脂合成中起重要作用。

Lysophosphatidic acid acyltransferase（LPAT） is a key enzyme in biosynthesis pathway of vegetable oil in plant.It is important for oil crops to improve oil quality and increase seed oil content through genetic engineering.We constructed a full-length cDNA library of peanut（Arachis hypogaea） seed via a large number of sequences of expressed sequence tag（EST） and gene functional annotation,a lysophosphatidic acid acyltransferase gene,designated AhLPAT,and its genomic DNA sequence were isolated from peanut.The sequence of AhLPAT cDNA was 1 629 bp,and its genomic sequence was 5 331 bp.Bioinformatic analysis showed that AhLPAT was composed of 11 exons and 10 introns with typical GT-AG characteristic in comparison of its sequences of genomic DNA and cDNA by Splign in NCBI.A peptide of 387 amino acid residues with protein molecular weight of 43.2 kD and isoelectric point（pI） of 9.42 were deduced from AhLPAT.Conserved domains prediction indicated that AhLPAT comprised a typical conserved acyltransferase domain and a conserved lysophospholipid acyltransferases domain.The deduced amino acid had a high identity with the LPAT proteins reported from other species.Amino acid similarities of LPAT protein be-tween peanut and Tropaeolum majus,Brassica napus,Crambe hispanica subsp.abyssinica,Ricinus communis,and Arabidopsis thaliana were 90%,89%,89%,88%,and 87%,respectively.A phylogenetic tree was constructed by the Neighbor-Joining method using MEGA5.0.The phylogenetic tree suggested that AhLPAT and AtLPAT2 derived from Arabidopsis thaliana were grouped into the same class.Both AhLPAT and AtLPAT2 were endoplasmic reticulum type LPATs.The tissue specific expression analysis by using quantitative RT-PCR assays indicated that AhLPAT was ubiquitously expressed in root,stem,leaf,flower,gynophore,seed of peanut with the highest level in gynophore and seed.The expression level reached a peak in the stage from 50 to 60 days after flowering.The expression level of AhLPAT kept consistent with the rate of oil accumulation,indicating a significant correla-tion between AhLPAT expression level and oil content（r=0.63,P0.05）.These results suggest that AhLPAT plays an important role in peanut triacylglycerol synthesis.