欢迎访问作物学报，今天是
作物学报

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

PDF

1967

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

[1]Ohlrogge J, Browse J. Lipid biosynthesis. Plant Cell, 1993, 7: 957–970

[2]Maisonneuve S, Bessoule J J, Lessire R, Delseny M, Roscoe T J. Expression of rapeseed microsomal lysophosphatidic acid acyltransferase isozymes enhances seed oil content in Arabidopsis. Plant Physiol, 2010, 152: 670–684

[3]Kennedy E. Sailing to Byzantlum. Annu Rev Biochem, 1992, 61: 1–8

[4]Weselake R J, Taylor D C, Rahman M H, Shah S, Laroche A, McVetty P B, Harwood J L. Increasing the flow of carbon into seed oil. Biotechnol Adv, 2009, 27: 866–878

[5]Kim H U, Li Y, Huang A H C. Ubiquitous and endoplasmic reticulum–located lysophosphatidyl acyltransferase, LPAT2, is essential for female but not male gametophyte development in Arabidopsis. Plant Cell, 2005, 17: 1073–1089

[6]Bourgis F, Kader J C, Barret P, Renard M, Robinson D, Robinson C, Delseny M, Roscoe T J. A plastidial lysophosphatidic acid acyltransferase from oilseed rape. Plant Physiol, 1999, 120: 913–921

[7]Knutzon D S, Lardizabal K D, Nelsen J S, Bleibaum J L, Davies H M, Metz J C. Cloning of a coconut endosperm cDNA encoding a 1-acyl-sn-glycerol-3-phosphate acyltransferase that accepts medium-chain-length substrates. Plant Physiol, 1995, 109: 999–1006

[8]Brown A P, Coleman J, Tommey A M, Watson M D, Slabas A R. Isolation and characterization of a maize cDNA that complements a 1-acyl-sn-glycerol-3-phosphate acyltaransferase mutant of Escherichia coli and encodes a protein which similarities to other acyltransferases. Plant Mol Biol, 1994, 26: 211–223

[9]Taylor D C, Francis T, Lozinsky S, Hoffman T, Giblin M, Marillia E F. Cloning and characterization of a constitutive lysophosphatidic acid acyltransferase 2 (LPAT2) gene from Tropaeolum majus L. Open Plant Sci J, 2010, 4: 7–17

[10]Roscoe T J. Identification of acyltransferases controlling triacylglycerol biosynthesis in oilseeds using a genomics-based approach. Eur J Lipid Sci Technol, 2005, 107: 256–262

[11]Hares W, Frentzen M. Substrate specificities of the membrane-bound and partially purified microsomal acyl-CoA: 1-acylglycerol-3-phosphate acyltransferase from etiolated shoots of Pisum satirum (L.). Planta, 1991, 185: 124–131

[12]Kim H U, Huang A H C. Plastid lysophosphatidyl acytransferase is essential for embryo development in Arabidopsis. Plant Physiol, 2004, 134: 1206–1216

[13]Löhden I, Frentzen M. Triacylglycerol biosynthesis in developing seeds of Tropaeolum majus L. and Limnanthes douglasii R. Br. Planta, 1992, 188: 215–224

[14]Bernertha R, Frentzena M. Utilization of erucoyl-CoA by acyltransferases from developing seeds of Brassica napus (L.) involved in triacylglycerol biosynthesis. Plant Sci, 1990, 67: 21–28

[15]Somerville C R, Browse J, Jaworski J G, Ohlrogge J B. Lipids. In: Buchanan B B, Gruissem W, Jones R L, eds. Biochemistry and Molecular Biology of Plants. Rockville, MD: American Society of Plant Physiologists, 2000. pp 456–527

[16]Zou J, Katavic V, Giblin E M, Barton D L, MacKenzie S L, Keller W A, Hu X, Taylor D C. Modification of seed oil content and acyl composition in the brassicaceae by expression of a yeast sn-2 acyltransferase gene. Plant Cell, 1997, 9: 909–923

[17]Lavia G I, Fernández A. Genome size in wild and cultivated peanut germplasm. Plant Syst Evol, 2008, 272: 1–10

[18]Kapustin Y, Souvorov A, Tatusova T, Lipman D. Splign: algorithms for computing spliced alignments with identification of paralogs. Biology Direct, 2008, 3:20

[19]Marchler-Bauer A, Lu S, Anderson J B, Chitsaz F, Derbyshire M K, DeWeese-Scott C, Fong J H, Geer L Y, Geer R C, Gonzales N R, Gwadz M, Hurwitz D I, Jackson J D, Ke Z, Lanczycki C J, Lu F, Marchler G H, Mullokandov M, Omelchenko M V, Robertson C L, Song J S, Thanki N, Yamashita R A, Zhang D, Zhang N, Zheng C, Bryant S H. CDD: a conserved domain database for the functional annotation of proteins. Nucl Acids Res, 2011, 39: 225–229

[20]Luo M, Dang P, Bausher B M, Holbrook C C, Lee R D, Lynch R E, Guo B Z. Identification of transcripts involved in resistance responses to leaf spot disease caused by Cercosporidium personatum in peanut (Arachis hypogaea). Phytopathological, 2005, 95: 381–387

[21]Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods, 2001, 25: 402–408

[22]Yu B, Wakao S, Fan J, Benning C. Loss of plastic lysophosphatidic acid acyltransferase cause embryo-lethality in Arabidopsis. Plant Cell Physiol, 2004, 45: 503–510

[23]Frentzen M. Biosynthesis and desaturation of the different diacylglycerol moieties in higher plants. Plant Physiol, 1986, 124: 193–209

[24]Lewin T M, Wang P, Coleman R A. Analysis of amino acid motifs diagnostic for the sn-glycerol-3-phosphate acyltransferase reaction. Biochemistry, 1999, 38: 5764–5771

[25]Heath R J, Rock C O. A conserved histidine is essential for glycerolipid acyltransferase catalysis. J Bacteriol, 1998, 180: 1425–1430

[26]Leung D W. The structure and functions of human lysophosphatidic acid acyltransferases. Frontiers in Bioscience, 2001, 6: 944–953

[27]Bennett M D, Leitch I J. Nuclear DNA amounts in angiosperms. Ann Bot, 1995, 76: 113–176

[28]Guo B, Chen X, Dang P, Scully B T, Liang X, Holbrook C C, Yu J, Culbreath A K. Peanut gene expression profiling in developing seeds at different reproduction stages during Aspergillus parasiticus infection. BMC Dev Biol, 2008, 8:12

[29]Cao Y Z, Oo K C, Huang A H C. Lysophosphatidate acyltransferase in the microsomes from maturing seeds of meadowfoam (Limnanthes alba). Plant Physiol, 1990, 94: 1199–1206

[30]Laurant P, Huang A H C. Organ and development specific acyl CoA lysophosphatidate acyltransferase in palm and meadowfoam. Plant Physiol, 1992, 99: 1711–1715

[31]Chen S-L(陈四龙), Li Y-R(李玉荣), Xu G-Z(徐桂真), Cheng Z-S(程增书). Simulation on oil accumulation characteristics in different high-oil peanut varieties. Acta Agron Sin (作物学报), 2008, 34(1): 142−149 (in Chinese with English abstract)

[32]Beisson F, Koo A J, Ruuska S, Schwender J, Pollard M, Thelen J J, Paddock T, Salas J J, Savage L, Milcamps A, Mhaske V B, Cho Y, Ohlrogge J B. Arabidopsis genes in-volved in acyl lipid metabolism. A 2003 census of the candi-dates, a study of the distribution of expressed sequence tags in organs, and a web-based database. Plant Physiol, 2003, 132: 681–697

[33]Bi Y P, Liu W, Xia H, Su L, Zhao C Z, Wan S B, Wang X J. EST sequencing and gene expression profiling of cultivated peanut (Arachis hypogaea L.). Genome, 2010, 53: 832–839

[34]Payton P, Kottapalli K R, Rowland R, Faircloth W, Guo B, Burow M, Puppala N, Gallo M. Gene expression profiling in peanut using high density oligonucleotide microarrays. BMC genomics, 2009, 10: 265

[35]Zhu S Q, Zhao H, Zhou R, Ji B H, Dan X Y. Substrate selectivity of glycerol-3-phosphate acyl transferase in rice. J Integr Plant Biol, 2009, 51: 1040–1049

[36]Sun C, Cao Y Z, Huang A H C. Acyl Coenzyme A preference of the glycerol phosphate pathway in the microsomes from the maturing seeds of palm, maize, and rapeseed. Plant Physiol, 1988, 88: 56–60
[1] 崔连花, 詹为民, 杨陆浩, 王少瓷, 马文奇, 姜良良, 张艳培, 杨建平, 杨青华. 2个玉米ZmCOP1基因的克隆及其转录丰度对不同光质处理的响应[J]. 作物学报, 2022, 48(6): 1312-1324.
[2] 陈松余, 丁一娟, 孙峻溟, 黄登文, 杨楠, 代雨涵, 万华方, 钱伟. 甘蓝型油菜BnCNGC基因家族鉴定及其在核盘菌侵染和PEG处理下的表达特性分析[J]. 作物学报, 2022, 48(6): 1357-1371.
[3] 周慧文, 丘立杭, 黄杏, 李强, 陈荣发, 范业赓, 罗含敏, 闫海锋, 翁梦苓, 周忠凤, 吴建明. 甘蔗赤霉素氧化酶基因ScGA20ox1的克隆及功能分析[J]. 作物学报, 2022, 48(4): 1017-1026.
[4] 晋敏姗, 曲瑞芳, 李红英, 韩彦卿, 马芳芳, 韩渊怀, 邢国芳. 谷子糖转运蛋白基因SiSTPs的鉴定及其参与谷子抗逆胁迫响应的研究[J]. 作物学报, 2022, 48(4): 825-839.
[5] 靳容, 蒋薇, 刘明, 赵鹏, 张强强, 李铁鑫, 王丹凤, 范文静, 张爱君, 唐忠厚. 甘薯Dof基因家族挖掘及表达分析[J]. 作物学报, 2022, 48(3): 608-623.
[6] 谢琴琴, 左同鸿, 胡燈科, 刘倩莹, 张以忠, 张贺翠, 曾文艺, 袁崇墨, 朱利泉. 甘蓝自交不亲和相关基因BoPUB9的克隆及表达分析[J]. 作物学报, 2022, 48(1): 108-120.
[7] 尹明, 杨大为, 唐慧娟, 潘根, 李德芳, 赵立宁, 黄思齐. 大麻GRAS转录因子家族的全基因组鉴定及镉胁迫下表达分析[J]. 作物学报, 2021, 47(6): 1054-1069.
[8] 许静, 潘丽娟, 李昊远, 王通, 陈娜, 陈明娜, 王冕, 禹山林, 侯艳华, 迟晓元. 花生油脂合成相关基因的表达谱分析[J]. 作物学报, 2021, 47(6): 1124-1137.
[9] 唐锐敏, 贾小云, 朱文娇, 印敬明, 杨清. 马铃薯热激转录因子HsfA3基因的克隆及其耐热性功能分析[J]. 作物学报, 2021, 47(4): 672-683.
[10] 李鹏程, 毕真真, 孙超, 秦天元, 梁文君, 王一好, 许德蓉, 刘玉汇, 张俊莲, 白江平. DNA甲基化参与调控马铃薯响应干旱胁迫的关键基因挖掘[J]. 作物学报, 2021, 47(4): 599-612.
[11] 贾小平, 李剑峰, 张博, 全建章, 王永芳, 赵渊, 张小梅, 王振山, 桑璐曼, 董志平. 谷子SiPRR37基因对光温、非生物胁迫的响应特点及其有利等位变异鉴定[J]. 作物学报, 2021, 47(4): 638-649.
[12] 岳洁茹, 白建芳, 张风廷, 郭丽萍, 苑少华, 李艳梅, 张胜全, 赵昌平, 张立平. 杂交小麦抗坏血酸过氧化物酶基因克隆及其在种子老化中的潜在功能分析[J]. 作物学报, 2021, 47(3): 405-415.
[13] 牛娜, 刘震, 黄鹏翔, 朱金勇, 李志涛, 马文婧, 张俊莲, 白江平, 刘玉汇. 马铃薯GAUT基因家族的全基因组鉴定及表达分析[J]. 作物学报, 2021, 47(12): 2348-2361.
[14] 解盼, 刘蔚, 康郁, 华玮, 钱论文, 官春云, 何昕. 甘蓝型油菜CBF基因家族的鉴定和表达分析[J]. 作物学报, 2021, 47(12): 2394-2406.
[15] 杨琴莉, 杨多凤, 丁林云, 赵汀, 张军, 梅欢, 黄楚珺, 高阳, 叶莉, 高梦涛, 严孙艺, 张天真, 胡艳. 棉花花器官突变体的鉴定及候选基因的克隆[J]. 作物学报, 2021, 47(10): 1854-1862.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备15008789号-2