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作物学报 ›› 2012, Vol. 38 ›› Issue (02): 245-255.doi: 10.3724/SP.J.1006.2012.00245

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

花生溶血磷脂酸酰基转移酶基因的克隆与表达分析

陈四龙1,2，黄家权1，雷永1，任小平1，文奇根1，陈玉宁1，姜慧芳1，晏立英1，廖伯寿1,*   

  1. 1 中国农业科学院油料作物研究所 / 农业部油料作物生物学重点开放实验室, 湖北武汉 430062; 2 河北省农林科学院粮油作物研究所 / 河北省作物遗传育种实验室, 河北石家庄 050031
  • 收稿日期:2011-05-26 修回日期:2011-09-13 出版日期:2012-02-12 网络出版日期:2011-12-01
  • 通讯作者: 廖伯寿, E-mail: lboshou@hotmail.com, Tel: 027-86812725
  • 基金资助:

    本研究由河北省自然科学基金项目(C2010001594), 国家自然科学基金项目(31071456), 现代农业产业技术体系建设专项资金(nycytx-19), 河北省重点基础研究项目(10960122D)和中国农业科学院油料作物研究所中央级公益性科研院所基本科研业务费专项(1610172010001)资助。

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

CHEN Si-Long1,2,HUANG Jia-Quan1,LEI Yong1,REN Xiao-Ping1,WEN Qi-Gen1,CHEN Yu-Ning1,JIANG Hui-Fang1,YAN Li-Ying1,LIAO Bo-Shou1,*   

  1. 1 Key Laboratory of Oil Crop Biology of the Ministry of Agriculture, Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan 430062, China; 2 Institute of Food and Oil Crops, Hebei Academy of Agriculture and Forestry Sciences, Laboratory of Crop Genetics and Breeding of Hebei Province, Shijiazhuang 050031, China
  • Received:2011-05-26 Revised:2011-09-13 Published:2012-02-12 Published online:2011-12-01
  • Contact: 廖伯寿, E-mail: lboshou@hotmail.com, Tel: 027-86812725

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被引次数

17

摘要： 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1 629 bp，对应的基因组序列5 531 bp，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43.2 kD，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50~60 d，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。

关键词: 转移酶, 基因克隆, 表达分析, 油脂合成

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 eleven exons and ten 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 between 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 Neighbour-Joining method using MEGA5.0. The phylogenetic tree suggested that AhLPAT and AtLPAT2 derived from Arabidopsis thaliana were grouped into the same class and 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 and seed of peanut with the highest level in gynephore 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 correlation between AhLPAT expression level and oil content (r=0.63, P<0.05). These results suggested that AhLPAT plays an important role in peanut triacylglycerol synthesis.

Key words: Peanut, Lysophosphatidic acid acyltransferase, Gene cloning, Expression analysis, Triacylglycerol synthesis

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