花生温度诱导载脂蛋白基因AhTIL1的克隆和表达研究
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摘要
温度诱导的载脂蛋白(temperature induced lipocalins,TILs)与植物在热、冷、光、氧化等胁迫下的稳定性相关,旨在克隆花生中的温度诱导的载脂蛋白基因,分析其在响应低温、高温中的作用。从花生c DNA文库中筛选到一个TIL候选基因,命名为Ah TIL1,通过PCR扩增获得了其基因组和候选启动子的序列,并利用实时定量PCR等方法检测了该基因在花生不同组织、种子不同发育时期和温度胁迫下的相对表达量。序列分析结果表明,该基因的开放阅读框(open reading frame,ORF)为558 bp,编码185个氨基酸。生物信息学分析显示,推测的Ah TIL1蛋白质相对分子质量为21.4 k D,等电点为6.78。序列比对结果显示,该蛋白氨基酸序列同其他物种TIL蛋白显示出很高的同源性。对候选启动子预测结果表明,该基因的候选启动子包含ACE、Box4、G-box、GAG-motif等与光反应相关的调控元件以及与ABA、水杨酸、赤霉素、厌氧等相关的顺式调控元件。基因芯片和RNASeq结果表明,该基因在花生的根、茎、叶、花、果针、种子中均有表达,在花中表达量最高,茎中次之,在根中表达量最低;Ah TIL1在果针入土后表达量下降,随着荚果的膨大和种子的成熟,Ah TIL1的表达量逐渐增加。实时定量PCR结果证明,Ah TIL1在高温和低温诱导3、6、12和24 h后上调表达,在48 h后表达量下降。Ah TIL1可能在花生响应低温、冷胁迫中发挥着重要的作用。
The temperature-induced lipocalins(TILs)is correlated with the stability of plant under stress environment of hot,cold,lighting,and oxidizing. This study intended to clone the TIL gene from peanut and analyze its function in response to temperature stress. A gene encoding TIL,designated as Ah TIL1,was screened from the peanut c DNA library. The relative expressions of the gene in the different tissues of peanuts,at the different stages of seeds,and under the temperature stress were detected by quantitative PCR. The results of sequence analysis showed that the length of the ORF of Ah TIL1 was 558 bp,encoding 185 amino acids. The bioinformatics analysis indicated that the predicted molecular mass of encoded protein was 21.4 k D,ad p I was 6.78. Sequence alignment displayed that the amino acids of Ah TIL1 were in high homology with TIL of other species. The predicted results of candidate promoter showed that the candidate gene promoter contained regulatory elements associated with the light reaction such as ACE,Box4,G-box,GAG-motif etc.,and other related cis regulatory elements such as ABA,salicylic acid,gibberellin,anaerobic etc. Microarray and RNA-seq results showed that Ah TIL1 expressed in roots,stems,leaves,flowers,peg,and seeds,the highest in the flowers,second highest in the stems,and the least in the roots. After the peg penetrating into soil,the expression level of Ah TIL1 decreased immediately,and then increased gradually with the enlarging and maturing of seeds. Real time quantitative PCR result proved that the expression level of Ah TIL1 increased after low or high temperature treatment for 3,6,12 and 24 h,however,decreased after 48 h. These results indicated that Ah TIL1 may play an important role in peanut response to temperature stress.
The temperature-induced lipocalins(TILs)is correlated with the stability of plant under stress environment of hot,cold,lighting,and oxidizing. This study intended to clone the TIL gene from peanut and analyze its function in response to temperature stress. A gene encoding TIL,designated as Ah TIL1,was screened from the peanut c DNA library. The relative expressions of the gene in the different tissues of peanuts,at the different stages of seeds,and under the temperature stress were detected by quantitative PCR. The results of sequence analysis showed that the length of the ORF of Ah TIL1 was 558 bp,encoding 185 amino acids. The bioinformatics analysis indicated that the predicted molecular mass of encoded protein was 21.4 k D,ad p I was 6.78. Sequence alignment displayed that the amino acids of Ah TIL1 were in high homology with TIL of other species. The predicted results of candidate promoter showed that the candidate gene promoter contained regulatory elements associated with the light reaction such as ACE,Box4,G-box,GAG-motif etc.,and other related cis regulatory elements such as ABA,salicylic acid,gibberellin,anaerobic etc. Microarray and RNA-seq results showed that Ah TIL1 expressed in roots,stems,leaves,flowers,peg,and seeds,the highest in the flowers,second highest in the stems,and the least in the roots. After the peg penetrating into soil,the expression level of Ah TIL1 decreased immediately,and then increased gradually with the enlarging and maturing of seeds. Real time quantitative PCR result proved that the expression level of Ah TIL1 increased after low or high temperature treatment for 3,6,12 and 24 h,however,decreased after 48 h. These results indicated that Ah TIL1 may play an important role in peanut response to temperature stress.
引文
[1]严美玲,李向东,林英杰,等.苗期干旱胁迫对不同抗旱花生品种生理特性、产量和品质的影响[J].作物学报,2007,33(1):113-119.
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[11]Charron JB,Ouellet F,Houde M,et al.The plant Apolipoprotein D ortholog protects Arabidopsis against oxidative stress[J].BMC Plant Biol,2008,8(1):86.
[12]Abo-Ogiala A,Carsjens C,Diekmann H,et al.Temperatureinduced lipocalin(TIL)is translocated under salt stress and protects chloroplasts from ion toxicity[J].Plant Physiology,2014,171(3-4):250-259.
[13]Levesque-Tremblay G,Havaux M,Ouellet F.The chloroplastic lipocalin At CHL prevents lipid peroxidation and protects Arabidopsis against oxidative stress[J].Plant J,2009,60(4):691-702.
[14]Boca S,Koestler F,Ksas B,et al.Arabidopsis lipocalins At CHL and At TIL have distinct but overlapping functions essential for lipid protection and seed longevity[J].Plant,Cell&Environment,2014,37(2):368-381.
[15]全先庆,高成香,王兴军,等.花生金属硫蛋白基因Ah MT3a的克隆及其表达[J].生物技术通报,2012(3):75-79.
[16]Bishop RE.The bacterial lipocalins[J].Biochim Biophys Acta,2000,1482(1-2):73-83.
[17]Fluckinger M,Haas H,Merschak P,et al.Human tear lipocalin exhibits antimicrobial activity by scavenging microbial siderophores[J].Antimicrob Agents Chemother,2004,48(9):3367-3372.
[18]Ganfornina MD,Do Carmo S,Lora JM,et al.Apolipoprotein D is involved in the mechanisms regulating protection from oxidative stress[J].Aging Cell,2008,7(4):506-515.
[19]Burow MD,Simpson CE,Faries MW,et al.Molecular biogeographic study of recently described B-and A-genome Arachis species,also providing new insights into the origins of cultivated peanut[J].Genome,2009,52(2):107-119.
[20]Su L,Zhao C,Bi Y,et al.Isolation and expression analysis of LEA genes in peanut(Arachis hypogaea L.)[J].Journal of Biosciences,2011,36(2):223-228.
[21]苏磊,姜娜娜,王兴军,等.花生Ah LEA18蛋白基因的克隆与表达分析[J].中国农学通报,2010,26(17):47-50.
[22]朱岩,彭振英,范仲学,等.花生蛋白激酶基因Ah STK的克隆与序列分析[J].山东农业科学,2013,45(1):9-13.
[23]刘羽,赵传志,李长生,等.花生HIR基因的克隆表达与进化分析[J].山东农业科学,2014,46(5):1-6.
[2]宰学明,钦佩,吴国荣,等.高温胁迫对花生幼苗光合速率、叶绿素含量、叶绿体Ca2+-ATPase、Mg2+-ATPase及Ca2+分布的影响[J].植物研究,2007,27(4):416-420.
[3]陈建爱,郭峰,杨武汉,等.盐胁迫下黄绿木霉T1010对花生耐盐生理的影响[J].西南农业学报,2014,27(2):587-590.
[4]Sanchez D.Exon-intron structure and evolution of the lipocalin gene family[J].Molecular Biology and Evolution,2003,20(5):775-783.
[5]Grzyb J,Latowski D,Strza?ka K.Lipocalins-a family portrait[J].Journal of Plant Physiology,2006,163(9):895-915.
[6]Flower DR,North AC,Sansom CE.The lipocalin protein family:structural and sequence overview[J].Biochim Biophys Acta,2000,1482(1-2):9-24.
[7]Frenette CJ,Breton G,Badawi M,et al.Molecular and structural analyses of a novel temperature stress-induced lipocalin from wheat and Arabidopsis[J].FEBS Lett,2002,517(1-3):129-132.
[8]Chi WT,Fung RW,Liu HC,et al.Temperature-induced lipocalin is required for basal and acquired thermo tolerance in Arabidopsis[J].Plant Cell Environ,2009,32(7):917-927.
[9]Kawamura Y,Uemura M.Mass spectrometric approach for identifying putative plasma membrane proteins of Arabidopsis leaves associated with cold acclimation[J].Plant J,2003,36(2):141-154.
[10]Charron JB,Ouellet F,Pelletier M,et al.Identification,expression,and evolutionary analyses of plant lipocalins[J].Plant Physiol,2005,139(4):2017-2028.
[11]Charron JB,Ouellet F,Houde M,et al.The plant Apolipoprotein D ortholog protects Arabidopsis against oxidative stress[J].BMC Plant Biol,2008,8(1):86.
[12]Abo-Ogiala A,Carsjens C,Diekmann H,et al.Temperatureinduced lipocalin(TIL)is translocated under salt stress and protects chloroplasts from ion toxicity[J].Plant Physiology,2014,171(3-4):250-259.
[13]Levesque-Tremblay G,Havaux M,Ouellet F.The chloroplastic lipocalin At CHL prevents lipid peroxidation and protects Arabidopsis against oxidative stress[J].Plant J,2009,60(4):691-702.
[14]Boca S,Koestler F,Ksas B,et al.Arabidopsis lipocalins At CHL and At TIL have distinct but overlapping functions essential for lipid protection and seed longevity[J].Plant,Cell&Environment,2014,37(2):368-381.
[15]全先庆,高成香,王兴军,等.花生金属硫蛋白基因Ah MT3a的克隆及其表达[J].生物技术通报,2012(3):75-79.
[16]Bishop RE.The bacterial lipocalins[J].Biochim Biophys Acta,2000,1482(1-2):73-83.
[17]Fluckinger M,Haas H,Merschak P,et al.Human tear lipocalin exhibits antimicrobial activity by scavenging microbial siderophores[J].Antimicrob Agents Chemother,2004,48(9):3367-3372.
[18]Ganfornina MD,Do Carmo S,Lora JM,et al.Apolipoprotein D is involved in the mechanisms regulating protection from oxidative stress[J].Aging Cell,2008,7(4):506-515.
[19]Burow MD,Simpson CE,Faries MW,et al.Molecular biogeographic study of recently described B-and A-genome Arachis species,also providing new insights into the origins of cultivated peanut[J].Genome,2009,52(2):107-119.
[20]Su L,Zhao C,Bi Y,et al.Isolation and expression analysis of LEA genes in peanut(Arachis hypogaea L.)[J].Journal of Biosciences,2011,36(2):223-228.
[21]苏磊,姜娜娜,王兴军,等.花生Ah LEA18蛋白基因的克隆与表达分析[J].中国农学通报,2010,26(17):47-50.
[22]朱岩,彭振英,范仲学,等.花生蛋白激酶基因Ah STK的克隆与序列分析[J].山东农业科学,2013,45(1):9-13.
[23]刘羽,赵传志,李长生,等.花生HIR基因的克隆表达与进化分析[J].山东农业科学,2014,46(5):1-6.