小麦TaCP3基因的克隆及其参与非生物胁迫的表达分析
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摘要
半胱氨酸蛋白酶(cysteine protease,CP)是植物中重要的蛋白酶家族之一,广泛参与植物的各种生理过程;TaCP3属于papain-like(木瓜蛋白酶)家族的半胱氨酸蛋白酶,在小麦的生长发育及抗逆中起到重要作用。为研究 TaCP3基因的结构特征,以及在干旱、高盐、低温和高温胁迫下的表达情况,从小麦抗旱品种西农538中克隆了 TaCP3基因,该基因仅含1个1125 bp的开放阅读框,编码374个氨基酸,在蛋白氨基端有一个28个氨基酸残基组成的信号肽,羧基端具有木瓜蛋白酶亚家族的保守结构域。生物信息学分析表明, TaCP3与大麦和山羊草半胱氨酸蛋白酶基因相似性最高。同时,本研究构建了pcold-TF/TaCP3原核表达载体,通过转化大肠杆菌BL21(DE3),成功表达了TaCP3重组蛋白,分子量约为40 kD。qRT-PCR结果表明, TaCP3的表达对干旱、高盐、低温和高温胁迫均有响应,初始都呈先降后升的趋势;且对干旱胁迫有强烈的正向响应。
Cysteine protease(CP) is one of the most important protease families in plants.It is widely involved in various physiological processes of plants. TaCP3 belongs to the papain-like family of the cysteine protease.It also plays an important role in wheat growth and development and stress resistance.In order to study the structural characteristics of TaCP3 gene and its expression under drought stress,high salt stress,low and high temperature stresses,the TaCP3 gene was cloned from the common wheat variety Xinong 538.The gene contains only a 1 125 bp open reading frame and encodes a protein containing 374 amino acids.There is a signal peptide consisting of 28 amino acid residues at the N-terminus of the protein,and the C-terminus has a conserved domain of the papain subfamily.Bioinformatics analysis showed that TaCP3 had the highest similarity with cysteine protease genes in barley and Aegilops.We constructed the pcold-TF/TaCP3 prokaryotic expression vector and successfully expressed TaCP3 recombinant protein by transforming E.coli BL21(DE3) with molecular weight of approximately 40 kD.The results of qRT-PCR showed that the expression of TaCP3 was all responded to drought stress,high salt stress,low and high temperature stresses,and the initial changes showed a trend of 'first decrease and then increase'.There was a strong positive response under drought stress.
Cysteine protease(CP) is one of the most important protease families in plants.It is widely involved in various physiological processes of plants. TaCP3 belongs to the papain-like family of the cysteine protease.It also plays an important role in wheat growth and development and stress resistance.In order to study the structural characteristics of TaCP3 gene and its expression under drought stress,high salt stress,low and high temperature stresses,the TaCP3 gene was cloned from the common wheat variety Xinong 538.The gene contains only a 1 125 bp open reading frame and encodes a protein containing 374 amino acids.There is a signal peptide consisting of 28 amino acid residues at the N-terminus of the protein,and the C-terminus has a conserved domain of the papain subfamily.Bioinformatics analysis showed that TaCP3 had the highest similarity with cysteine protease genes in barley and Aegilops.We constructed the pcold-TF/TaCP3 prokaryotic expression vector and successfully expressed TaCP3 recombinant protein by transforming E.coli BL21(DE3) with molecular weight of approximately 40 kD.The results of qRT-PCR showed that the expression of TaCP3 was all responded to drought stress,high salt stress,low and high temperature stresses,and the initial changes showed a trend of 'first decrease and then increase'.There was a strong positive response under drought stress.
引文
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[13]马岩岩,张军,陈娇,等.柑橘半胱氨酸蛋白酶基因CsCysP的分离、亚细胞定位及表达分析[J].园艺学报,2014,41(4):621.MA Y Y,ZHANG J ,CHEN J,et al.Isolation,subcellular localization and expression analysis of a Citrus cysteine protease gene,CsCysP [J].Acta Horticulturae Sinica,2014,41(4):621.
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[16]臧庆伟.小麦抗旱相关基因 TaCP和 TaLTP1的克隆及功能分析[D].北京:中国农业科学院,2005:29.ZANG Q W.Cloning and functional analysis of drought-resistant genes TaCP and TaLTP1 in wheat [D].Beijing:Chinese Academy of Agricultural Sciences,2005:29.
[17]GUERRERO C,CALLE M,REID M S,et al.Analysis of the expression of two thioprotease genes from day lily(Hemerocallis spp.) during flower senescence [J].Plant Molecular Biology,1998,36:565.
[18]AO T G,LANG M L,LI Y Q,et al.Cloning and expression analysis of cysteine protease gene(MwCP) in Agropyron mongolicum Keng [J].Genetics and Molecular Research,2015,15(1) :14.
[19]JENNIFER T J,JOHN E M.A salt- and dehydration-inducible pea gene, Cyp15a,encodes a cell-wall protein with sequence similarity to cysteine protease [J].Plant Molecular Biology,1995,28:1055.
[20]方正武,李来运,李晓方,等.甜荞木瓜类半胱氨酸蛋白酶基因 FeRD21的克隆与表达分析(英文)[J].西北植物学报,2015,35(3):459.FANG Z W,LI Y L,LI X F,et al.Cloning and expression analysis of a papain-like cystein protease gene(FeRD21) in Fagopyrum esculentum(English) [J].Acta Botanica Boreali-Occidentalia Sinica,2015,35(3):459.
[21]GRUDKOWSKA M,ZAGDANSKA B.Acclimation to frost alters proteolytic response of wheat seedlings to drought [J].Journal of Plant Physiology,2010,167(16):1321.
[22]WINIEWSKI K,ZAGDASKA B.Genotype-dependent proteolytic response of spring wheat to water deficiency [J].Journal of Experimental Botany,2001,52(360):1455.
[2]BASSET G,RAYMOND P,MALEK L,et al.Changes in the expression and the enzymic properties of the 20S proteasome in sugar-starved maize roots.Evidence for an in vivo oxidation of the proteasome [J].Plant Physiology,2002,128(3):1149.
[3]闫龙凤,杨青川,韩建国,等.植物半胱氨酸蛋白酶研究进展[J].草业学报,2005,14(5):11.YAN L F,YANG Q C,HAN J G,et al.Research advances in plant cysteine proteinases [J].Acta Prataculturae Sinica,2005,14(5):11.
[4]BEERS E P,JONES A M,DICKERMAN A W.The S8 serine,C1A cysteine and A1 aspartic protease families in Arabidopsis [J].Phytochemistry,2004,65(1):43.
[5]WATANABE N,LAM E.Two Arabidopsis metacaspases AtMCP1b and AtMCP2b are arginine/lysine-specific cysteine proteases and activate apoptosis-like cell death in yeast [J].Journal of Biological Chemistry,2005,280(15):14691.
[6]NONG V H,BECKER C,MNTZ K.cDNA cloning for a putative cysteine proteinase from developing seeds of soybean [J].Biochimica et Biophysica Acta(BBA)-Gene Structure and Expression,1995,1261(3):435.
[7]ESTEBAN-GARCíA B,GARRIDO-CáRDENAS J A,ALONSO D L,et al.A distinct subfamily of papain-like cystein proteinases regulated by senescence and stresses in glycine max [J].Journal of Plant Physiology,2010,167(13):1101.
[8]BEYENE G,FOYER C H,KUNERT K J.Two new cysteine proteinases with specific expression patterns in mature and senescent tobacco(Nicotiana tabacum L.) leaves [J].Journal of Experimental Botany,2006,57(6):1431.
[9]LEE S,JUNG K H,AN G,et al.Isolation and characterization of a rice cysteine protease gene, OsCP1,using T-DNA gene-trap system [J].Plant Molecular Biology,2004,54(5):755.
[10]WIEDERANDERS B.Structure-function relationships in class CA1 cysteine peptidase propeptides [J].Acta Biochimica Polonica,2003,50(3):691.
[11]BEERS E P,WOFFENDEN B J,ZHAO C.Plant proteolytic enzymes:Possible roles during programmed cell death [J].Plant Molecular Biology,2000,44(3):399.
[12]ZANG Q W,WANG C X,LI X Y,et al.Isolation and characterization of a gene encoding a polyethylene glycol-induced cysteine protease in common wheat [J].Journal of Biosciences,2010,35(3):379.
[13]马岩岩,张军,陈娇,等.柑橘半胱氨酸蛋白酶基因CsCysP的分离、亚细胞定位及表达分析[J].园艺学报,2014,41(4):621.MA Y Y,ZHANG J ,CHEN J,et al.Isolation,subcellular localization and expression analysis of a Citrus cysteine protease gene,CsCysP [J].Acta Horticulturae Sinica,2014,41(4):621.
[14]MUKKAYM G.THOMPSN W F.Rapid isolation of high molecular mass plant DNA [J].Nucleic Acids Research,1980,8(19):4321.
[15]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 [J].Methods,2001,25(4):402.
[16]臧庆伟.小麦抗旱相关基因 TaCP和 TaLTP1的克隆及功能分析[D].北京:中国农业科学院,2005:29.ZANG Q W.Cloning and functional analysis of drought-resistant genes TaCP and TaLTP1 in wheat [D].Beijing:Chinese Academy of Agricultural Sciences,2005:29.
[17]GUERRERO C,CALLE M,REID M S,et al.Analysis of the expression of two thioprotease genes from day lily(Hemerocallis spp.) during flower senescence [J].Plant Molecular Biology,1998,36:565.
[18]AO T G,LANG M L,LI Y Q,et al.Cloning and expression analysis of cysteine protease gene(MwCP) in Agropyron mongolicum Keng [J].Genetics and Molecular Research,2015,15(1) :14.
[19]JENNIFER T J,JOHN E M.A salt- and dehydration-inducible pea gene, Cyp15a,encodes a cell-wall protein with sequence similarity to cysteine protease [J].Plant Molecular Biology,1995,28:1055.
[20]方正武,李来运,李晓方,等.甜荞木瓜类半胱氨酸蛋白酶基因 FeRD21的克隆与表达分析(英文)[J].西北植物学报,2015,35(3):459.FANG Z W,LI Y L,LI X F,et al.Cloning and expression analysis of a papain-like cystein protease gene(FeRD21) in Fagopyrum esculentum(English) [J].Acta Botanica Boreali-Occidentalia Sinica,2015,35(3):459.
[21]GRUDKOWSKA M,ZAGDANSKA B.Acclimation to frost alters proteolytic response of wheat seedlings to drought [J].Journal of Plant Physiology,2010,167(16):1321.
[22]WINIEWSKI K,ZAGDASKA B.Genotype-dependent proteolytic response of spring wheat to water deficiency [J].Journal of Experimental Botany,2001,52(360):1455.