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

Abstract

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.