割手密蛋白磷酸酶基因SsPP2C1的克隆及表达分析
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摘要
2C型丝氨酸/苏氨酸蛋白磷酸酶(PP2C)是植物体内脱落酸ABA信号传导关键负调控酶,在植物耐非生物胁迫中发挥着重要的作用。为探讨抗旱割手密材料中蛋白磷酸酶基因的序列及其表达特征,利用转录组测序数据,结合RT-PCR技术,克隆出编码割手密PP2C蛋白的基因,命名为SsPP2C1,其核苷酸序列编码区长855 bp,编码284个氨基酸残基,这些残基中包括了与二价金属离子结合的MED、DGH、DG和D结构域。系统发生分析结果表明,该氨基酸与谷子Sipp2c亲缘关系最近。实时荧光定量PCR分析结果表明在干旱协迫条件下,SsPP2C1基因主要在割手密的叶片中表达,在根和茎中的表达量相对较低;该基因在割手密伸长期表达量最高,成熟期次之,苗期最小;随着胁迫时间的延长,该基因的表达量显著升高,在胁迫6 d时达到最大值,为对照的4 940.27倍,随后显著下降,表明SsPP2c1基因与割手密抗旱性存在紧密关联。
Protein phosphatase PP2 C is an important negative regulator of abscisic acid ABA signal transduction in plants. and exerts crucial roles when plants are in abiotic stress. The cDNA of SsPP2C1 encoding protein phosphatase PP2C was cloned base on data of high-throughput transcriptome sequencing and real-time fluorescence quantitative analysis of drought-tolerant Saccharum spontaneum L.. The length of nucleotide sequence of this gene was 855 bp in length, encoding 284 amino acids residues, which included metal ions-binding domain such as MED, DGH, DG and D amino acid residues.. Phylogenetic tree results showed that SsPP2C1 was clustered closely with Sipp2c of Setaria italica. RT-PCR analysis showed that SsPP2C1 mainly expressed in S. spontaneumleaves,and its expression amount in the roots and stems was relatively low. The expression of this gene reached the highest at the elongation stage, followed by the mature stage, and the lowest at the seedling stage of S. spontaneum after drought stress. With the duration of drought stress, the expression of this gene increased significantly and reached its maximum at sixth days after stress with the highest expression amount of expression 4 940.27 times than that of un-treated material, then the expression amount of this gene declined remarkably, suggested that the SsPP2C1 gene might be closely related to drought resistance of S. spontaneum.
Protein phosphatase PP2 C is an important negative regulator of abscisic acid ABA signal transduction in plants. and exerts crucial roles when plants are in abiotic stress. The cDNA of SsPP2C1 encoding protein phosphatase PP2C was cloned base on data of high-throughput transcriptome sequencing and real-time fluorescence quantitative analysis of drought-tolerant Saccharum spontaneum L.. The length of nucleotide sequence of this gene was 855 bp in length, encoding 284 amino acids residues, which included metal ions-binding domain such as MED, DGH, DG and D amino acid residues.. Phylogenetic tree results showed that SsPP2C1 was clustered closely with Sipp2c of Setaria italica. RT-PCR analysis showed that SsPP2C1 mainly expressed in S. spontaneumleaves,and its expression amount in the roots and stems was relatively low. The expression of this gene reached the highest at the elongation stage, followed by the mature stage, and the lowest at the seedling stage of S. spontaneum after drought stress. With the duration of drought stress, the expression of this gene increased significantly and reached its maximum at sixth days after stress with the highest expression amount of expression 4 940.27 times than that of un-treated material, then the expression amount of this gene declined remarkably, suggested that the SsPP2C1 gene might be closely related to drought resistance of S. spontaneum.
引文
Arino J.,Casamayor A.,and Gonzalez A.,2011,Type 2C protein phosphatases in fungi,Eukaryotic Cell,10(1):21-33
Bremer G.,1961,Problems in breeding and cytology of sugar cane,Euphytica,12(1):178-188
Chen Y.Q.,Deng Z.H.,Guo C.F.,Chen R.H.,amd Zhang M.Q.,2007,Drought resistant evaluations of commonly used parents and their derived varieties,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),40(6):1108-1117(陈义强,邓祖湖,郭春芳,陈如凯,张木清,2007,甘蔗常用亲本及其衍生品种的抗旱性评价,中国农业科学,40(6):1108-1117)
Clark S.E.,Williams R.W.,and Meyerowitz E.M.,1997,The CLAVATAL1 gene encodes a putative receptor kinase that controls shoot and floral meristem size in Arabidopsis,Cell,89(4):575-585
Cohen P.,1989,The structure and regulation of protein phosphatases,Ann.Rev.Biochem.,58(1):453-508
Das A.K.,Helps N.R.,Cohen P.T.W.,and Barford D.,1996,Crystal structure of the protein serine/threonine phosphatase2C at 2.0 A resolution,Embo.J.,15(24):6798-6809
Gosti F.,Beaudoin N.,Seirzet C.,Webb A.A.R.,Vartaman N.,and Giarudat J.,1999,ABI1 Protein PhosPhatase 2C is a negative regulator of abscisic acid signaling,Plant Cell,11(10):1897-1910
Kerk D.,Bulgrien J.,Smith D.W.,Barsam B.,and Veretnik S.,2002,The complement of protein phosphatase catalytic subunits encoded in the genome of Arabidopsis,Plant Physiol.,129(2):908-925
Klumpp S.,Selke D.,and Hemresmeier J.,1998,Protein phosphatase type 2C active at physiological Mg2+:stimulation by unsaturated fatty acids,FEBS Lett.,437(3):229-232
Kuhn J.M.,Boisson dernier A.,Dizon M.B.,Maktabi M.H.,and Schroeder J.I.,2006,The protein phosphatase At PP2CAnegatively regulates abscisic acid signal transduction in Arabidopsis,and effects of abh1 on At PP2CA m RNA,Plant Physiol.,140:127-139
Li X.J.,Liu H.B.,Lin X.Q.,Wu Z.D.,Xu C.H.,and Liu X.L.,2015,In silico cloning and bioinformatics analysis of KNOXgene in sugarcane(Sckn1),Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(1):136-142(李旭娟,刘洪博,林秀琴,吴转娣,徐超华,刘新龙,2015,甘蔗KNOX基因(Sckn1)的电子克隆及生物信息学分析,基因组学与应用生物学,34(1):136-142)
Liu X.,Zhu Y.,Zhai H.,Cai H.,Ji W.,Luo X.,Li J.,and Bai X.,2012,At PP2CG1,a protein phosphatase 2C,positively regulates salt tolerance of Arabidopsis in abscisic acid-dependent manner,Biochem.Biophys.Res.Commun.,42(24):710-715
Livak K.J.,and Schmittgen T.D.,2001,Analysis of relative gene expression data using real-time quantitative PCR and the 2-△△CTmethod.,Methods,25(4):402-408
Lorenzo O.,Nicolas C.,Nicolas G.,and Rodriguez D.,2002,Molecular cloning of a functional protein phosphatase 2C(Fs PP2C)with unusual features and synergistically up-regulated by ABA and calcium in dormant seed s of Fagus sylvatica,Physiol.Plant,114(3):482-490
Lu G.,and Wang Y.,2008,Functional diversity of mammalian type 2C protein phosphatase isoforms:new tales from an old family,Clin.Exp.Pharmacol Physiol.,35(2):107-112
Mayda E.,Marques C.,Conejero V.,and Vera P.,1999,Expression of a pathogen induced gene can be mimicked by auxin insensitivity,Mol.Plant Microbe Interact.,13(1):23-31
Meskiene I.,Bogre L.,Glaser W.,Balog J.,Brandstotter M.,Zwergrer K.,Ammerer G.,and Hirt H.,1998,MP2C,a plant protein phosphatase 2C,functions as a negative regulator of mitogen-activated protein kinase pathways in yeast and plants,Proc.Natl.Acad.Sci.USA,95(4):1938-1943
Meskiere I.,Baudouin E.,Schweighofer A.,Liwosz A.,Onak C.,Rod-riguez P.L.,Jelinek H.,and Hirt H.,2003,Stress-induced protein phosphatase 2C is a negative regulator of a mitogen-activated protein kinase,J.Biol.Chem.,278(21):18945-18952
Nishiyama M.Y.J.,Ferreira S.S.,Tang P.Z.,Becker S.,Taliana A.,and Souza M.,2014,Full-length enriched cDNA libraries and ORFeome analysis of sugarcane hybrid and ancestor genotypes,PLoS One,9(9):e107351
Ohta M.,Guo Y.,Halfter U.,and Zhu J.K.,2003,A novel domain in the protein kinase SOS2 mediates interactionwith the protein phosphatase 2C ABI2,Proc.Natl.Acad.Sci.USA,100(20):11771-11776
Park J.W.,Benatti T.R.,Marconi T.,Yu Q.,Solisgracia N.,Mora V.,and Silva J.A.,2015,Cold responsive gene expression profiling of sugarcane and Saccharum spontaneum with functional analysis of a cold inducible Saccharum homolog of NOD26-like intrinsic protein to salt and water stress,PLoS One,10(5):51-56
Rodriguez P.L.,1998,Protein phosphatase 2C(PP2C)function in higher plants,Plant Mol.Biol.,38(6):919-927
Schweighofer A.,Hirt H.,Meskiene I.,2004,Plant PP2C phosphatases:emerging functions in stress signaling,Trends Plant Sci.,9(5):236-243
Sheng L.,2003,Protein phosphatases in plants,Ann.Rev.Plant Biol.,54(54):63-92
Singh A.,Giri J.,Kapoor S.,Tyagi A.K.,and Pandey G.K.,2010,Protein phosphatase complement in rice:genome-wide identification and transcriptional analysis under abiotic stress conditions and reproductive development,BMC Genomics,11(1):435-453
Sreeinivasan T.V.,Ahloowalia B.S.,and Heinz D.J.,eds.,1987,Cytogenetics,insugarcane improvement through breeding,Elsevier Press,Amsterdam,pp.211-253
Su J.,and Forchhammer K.,2013,Determinants for substrate specificity of the bacterial PP2C proteinphosphatase t PphAfrom Thermosynechococcus elongatus,FEBS J.,280(2):694-707
Tahtiharju S.,and Palva T.,2001,Antisense inhibition of protein phosphatase 2C accelerates cold acclimation in Arabidopsis thaliana,Plant J.,26(4):461-470
Vranova E.,Langebartels C.,van Montagu M.,Inze D.,and Camp W.V.,2000,Oxidative stress,heat shock and drought differentially affect expression of a tobacco protein phosphatase 2C,J.Exp.Bot.,51(351):l763-1764
Xiong L.,Schumaker K.S.,and Zhu J.K.,2002,Cell signaling during cold,drought,and salt stress,Plant Cell,14:S165-S183
Xue T.,Wang D.,Zhang S.,Ni F.,Jakab S.,Zheng C.,and Zhong Y.,2008,Genome-wide and expression analysis of protein phosphatase 2C in rice and Arabidopsis,BMC Genomics,9(1):550-571
Xu Y.F.,Li D.P.,Gu L.K.,Hu X.L.,Zong X.J.,and Li D.Q.,2005,Cloning and expression characteristics of protein phosphatase gene ZmPP2C of Zea mays roots,Zhiwu Shengli Yu Fenzi Shengwuxue Xuebao(Journal of Plant Physiology and Molecular Biology),3l(2):183-189(徐云峰,李大鹏,谷令坤,胡晓丽,宗晓娟,李德全,2005,玉米根系蛋白磷酸酶基因的克隆及表达特性,植物生理与分子生物学学报,3l(2):183-189)
Yao Y.L.,Xu L.,Hu X.W.,Xing S.L.,and Liu Y.,2016,The analysis of cloning and comparing of DREB2 group transcription factor genes from Saccharum spontaneum L.,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(9):2261-2267(姚艳丽,徐磊,胡小文,邢淑莲,刘洋,2016,割手密DREB2类转录因子的克隆与比较分析,分子植物育种,14(9):2261-2267)
Bremer G.,1961,Problems in breeding and cytology of sugar cane,Euphytica,12(1):178-188
Chen Y.Q.,Deng Z.H.,Guo C.F.,Chen R.H.,amd Zhang M.Q.,2007,Drought resistant evaluations of commonly used parents and their derived varieties,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),40(6):1108-1117(陈义强,邓祖湖,郭春芳,陈如凯,张木清,2007,甘蔗常用亲本及其衍生品种的抗旱性评价,中国农业科学,40(6):1108-1117)
Clark S.E.,Williams R.W.,and Meyerowitz E.M.,1997,The CLAVATAL1 gene encodes a putative receptor kinase that controls shoot and floral meristem size in Arabidopsis,Cell,89(4):575-585
Cohen P.,1989,The structure and regulation of protein phosphatases,Ann.Rev.Biochem.,58(1):453-508
Das A.K.,Helps N.R.,Cohen P.T.W.,and Barford D.,1996,Crystal structure of the protein serine/threonine phosphatase2C at 2.0 A resolution,Embo.J.,15(24):6798-6809
Gosti F.,Beaudoin N.,Seirzet C.,Webb A.A.R.,Vartaman N.,and Giarudat J.,1999,ABI1 Protein PhosPhatase 2C is a negative regulator of abscisic acid signaling,Plant Cell,11(10):1897-1910
Kerk D.,Bulgrien J.,Smith D.W.,Barsam B.,and Veretnik S.,2002,The complement of protein phosphatase catalytic subunits encoded in the genome of Arabidopsis,Plant Physiol.,129(2):908-925
Klumpp S.,Selke D.,and Hemresmeier J.,1998,Protein phosphatase type 2C active at physiological Mg2+:stimulation by unsaturated fatty acids,FEBS Lett.,437(3):229-232
Kuhn J.M.,Boisson dernier A.,Dizon M.B.,Maktabi M.H.,and Schroeder J.I.,2006,The protein phosphatase At PP2CAnegatively regulates abscisic acid signal transduction in Arabidopsis,and effects of abh1 on At PP2CA m RNA,Plant Physiol.,140:127-139
Li X.J.,Liu H.B.,Lin X.Q.,Wu Z.D.,Xu C.H.,and Liu X.L.,2015,In silico cloning and bioinformatics analysis of KNOXgene in sugarcane(Sckn1),Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(1):136-142(李旭娟,刘洪博,林秀琴,吴转娣,徐超华,刘新龙,2015,甘蔗KNOX基因(Sckn1)的电子克隆及生物信息学分析,基因组学与应用生物学,34(1):136-142)
Liu X.,Zhu Y.,Zhai H.,Cai H.,Ji W.,Luo X.,Li J.,and Bai X.,2012,At PP2CG1,a protein phosphatase 2C,positively regulates salt tolerance of Arabidopsis in abscisic acid-dependent manner,Biochem.Biophys.Res.Commun.,42(24):710-715
Livak K.J.,and Schmittgen T.D.,2001,Analysis of relative gene expression data using real-time quantitative PCR and the 2-△△CTmethod.,Methods,25(4):402-408
Lorenzo O.,Nicolas C.,Nicolas G.,and Rodriguez D.,2002,Molecular cloning of a functional protein phosphatase 2C(Fs PP2C)with unusual features and synergistically up-regulated by ABA and calcium in dormant seed s of Fagus sylvatica,Physiol.Plant,114(3):482-490
Lu G.,and Wang Y.,2008,Functional diversity of mammalian type 2C protein phosphatase isoforms:new tales from an old family,Clin.Exp.Pharmacol Physiol.,35(2):107-112
Mayda E.,Marques C.,Conejero V.,and Vera P.,1999,Expression of a pathogen induced gene can be mimicked by auxin insensitivity,Mol.Plant Microbe Interact.,13(1):23-31
Meskiene I.,Bogre L.,Glaser W.,Balog J.,Brandstotter M.,Zwergrer K.,Ammerer G.,and Hirt H.,1998,MP2C,a plant protein phosphatase 2C,functions as a negative regulator of mitogen-activated protein kinase pathways in yeast and plants,Proc.Natl.Acad.Sci.USA,95(4):1938-1943
Meskiere I.,Baudouin E.,Schweighofer A.,Liwosz A.,Onak C.,Rod-riguez P.L.,Jelinek H.,and Hirt H.,2003,Stress-induced protein phosphatase 2C is a negative regulator of a mitogen-activated protein kinase,J.Biol.Chem.,278(21):18945-18952
Nishiyama M.Y.J.,Ferreira S.S.,Tang P.Z.,Becker S.,Taliana A.,and Souza M.,2014,Full-length enriched cDNA libraries and ORFeome analysis of sugarcane hybrid and ancestor genotypes,PLoS One,9(9):e107351
Ohta M.,Guo Y.,Halfter U.,and Zhu J.K.,2003,A novel domain in the protein kinase SOS2 mediates interactionwith the protein phosphatase 2C ABI2,Proc.Natl.Acad.Sci.USA,100(20):11771-11776
Park J.W.,Benatti T.R.,Marconi T.,Yu Q.,Solisgracia N.,Mora V.,and Silva J.A.,2015,Cold responsive gene expression profiling of sugarcane and Saccharum spontaneum with functional analysis of a cold inducible Saccharum homolog of NOD26-like intrinsic protein to salt and water stress,PLoS One,10(5):51-56
Rodriguez P.L.,1998,Protein phosphatase 2C(PP2C)function in higher plants,Plant Mol.Biol.,38(6):919-927
Schweighofer A.,Hirt H.,Meskiene I.,2004,Plant PP2C phosphatases:emerging functions in stress signaling,Trends Plant Sci.,9(5):236-243
Sheng L.,2003,Protein phosphatases in plants,Ann.Rev.Plant Biol.,54(54):63-92
Singh A.,Giri J.,Kapoor S.,Tyagi A.K.,and Pandey G.K.,2010,Protein phosphatase complement in rice:genome-wide identification and transcriptional analysis under abiotic stress conditions and reproductive development,BMC Genomics,11(1):435-453
Sreeinivasan T.V.,Ahloowalia B.S.,and Heinz D.J.,eds.,1987,Cytogenetics,insugarcane improvement through breeding,Elsevier Press,Amsterdam,pp.211-253
Su J.,and Forchhammer K.,2013,Determinants for substrate specificity of the bacterial PP2C proteinphosphatase t PphAfrom Thermosynechococcus elongatus,FEBS J.,280(2):694-707
Tahtiharju S.,and Palva T.,2001,Antisense inhibition of protein phosphatase 2C accelerates cold acclimation in Arabidopsis thaliana,Plant J.,26(4):461-470
Vranova E.,Langebartels C.,van Montagu M.,Inze D.,and Camp W.V.,2000,Oxidative stress,heat shock and drought differentially affect expression of a tobacco protein phosphatase 2C,J.Exp.Bot.,51(351):l763-1764
Xiong L.,Schumaker K.S.,and Zhu J.K.,2002,Cell signaling during cold,drought,and salt stress,Plant Cell,14:S165-S183
Xue T.,Wang D.,Zhang S.,Ni F.,Jakab S.,Zheng C.,and Zhong Y.,2008,Genome-wide and expression analysis of protein phosphatase 2C in rice and Arabidopsis,BMC Genomics,9(1):550-571
Xu Y.F.,Li D.P.,Gu L.K.,Hu X.L.,Zong X.J.,and Li D.Q.,2005,Cloning and expression characteristics of protein phosphatase gene ZmPP2C of Zea mays roots,Zhiwu Shengli Yu Fenzi Shengwuxue Xuebao(Journal of Plant Physiology and Molecular Biology),3l(2):183-189(徐云峰,李大鹏,谷令坤,胡晓丽,宗晓娟,李德全,2005,玉米根系蛋白磷酸酶基因的克隆及表达特性,植物生理与分子生物学学报,3l(2):183-189)
Yao Y.L.,Xu L.,Hu X.W.,Xing S.L.,and Liu Y.,2016,The analysis of cloning and comparing of DREB2 group transcription factor genes from Saccharum spontaneum L.,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(9):2261-2267(姚艳丽,徐磊,胡小文,邢淑莲,刘洋,2016,割手密DREB2类转录因子的克隆与比较分析,分子植物育种,14(9):2261-2267)