杨树PeNHLd基因的克隆与表达模式分析
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摘要
含有NHL结构域的基因在人类、动物和微生物的生长发育过程中扮演着重要角色,但是在植物中关于该类基因的研究还未曾有过报道。为了探究该类基因在植物中的表达模式,本研究以"南林895"杨为材料,采用RACE技术克隆得到了PeNHLd基因,并通过RT-PCR对预测的ORF框进行了验证。杨树PeNHLd基因包含7个外显子和6个内含子,其c DNA全长为1 971 bp,包括1个1 449 bp的开放阅读框,可编码482个氨基酸,预测其蛋白质分子质量为53.9 kD,理论等电点7.24;该蛋白含有3个跨膜螺旋和2个保守的NHL结构域。实时定量PCR结果显示,Pe NHLd基因在干旱、高盐、激素、病原菌侵染等胁迫下有着明显的动态表达模式,对这些胁迫做出了迅速而强烈的响应;同时该基因在杨树根、茎、叶等不同组织器官中存在显著的表达差异,有着明显的组织特异性。本研究为探究PeNHLd基因在植物中的表达模式提供理论依据。
The NHL-containing genes play an important role in the growth and development of humans, animals and microbes, but these genes have not been reported in plants ever before. To explore the expression pattern of NHLd gene in plants, the Pe NHLd gene was cloned from the Populus clone "Nanlin895" using rapid amplification cDNA ends(RACE), and the predicted open reading frame(ORF) was verified by RT-PCR. The PeNHLd gene consisted of six introns and seven exons, its full-length cDNA is 1 971 bp with the open reading frame(ORF) of1 449 bp. It would encode for polypeptides of 482 amino acid residues with the molecular weight of 53.9 kD and the theoretical isoelectric point of 7.24. The Pe NHLd protein contains three transmembrane helices, and two conserved NHL domains. Real-time quantitative PCR results showed that PeNHLd gene had a strong dynamic expression pattern under drought, high salt, hormone and pathogen stress, and responded quickly and strongly to these stresses. At the same time, there were significant differences in the expression of the gene in different tissues and organs of poplar roots, stems and leaves, and had obvious tissue specificity. This study provided a theoretical basis for exploring the expression pattern of PeNHLd gene in plants.
The NHL-containing genes play an important role in the growth and development of humans, animals and microbes, but these genes have not been reported in plants ever before. To explore the expression pattern of NHLd gene in plants, the Pe NHLd gene was cloned from the Populus clone "Nanlin895" using rapid amplification cDNA ends(RACE), and the predicted open reading frame(ORF) was verified by RT-PCR. The PeNHLd gene consisted of six introns and seven exons, its full-length cDNA is 1 971 bp with the open reading frame(ORF) of1 449 bp. It would encode for polypeptides of 482 amino acid residues with the molecular weight of 53.9 kD and the theoretical isoelectric point of 7.24. The Pe NHLd protein contains three transmembrane helices, and two conserved NHL domains. Real-time quantitative PCR results showed that PeNHLd gene had a strong dynamic expression pattern under drought, high salt, hormone and pathogen stress, and responded quickly and strongly to these stresses. At the same time, there were significant differences in the expression of the gene in different tissues and organs of poplar roots, stems and leaves, and had obvious tissue specificity. This study provided a theoretical basis for exploring the expression pattern of PeNHLd gene in plants.
引文
Arama E.,Dickman D.,Kimchie Z.,Shearn A.,and Lev Z.,2000Mutations in the beta-propeller domain of the Drosophila brain tumor(brat)protein induce neoplasm in the larval brain Oncogene.,19(33):3706-3716
Bari R.,and Jones J.D.,2009,Role of plant hormones in plan defence responses,Plant Mol.Biol.,69(4):473-488
Buell C.R.,Joardar V.,Lindeberg M.,Selengut J.,Paulsen I.T.Gwinn M.L.,Dodson R.J.,Deboy R.T.,Durkin A.S.,Kolonay J.F.,Madupu R.,Daugherty S.,Brinkac L.,Beanan M.J.Haft D.H.,Nelson W.C.,Davidsen T.,Zafar N.,Zhou L.W.Liu J.,Yuan Q.P.,Khouri H.,Fedorova N.,Tran B.,Russel D.,Berry K.,Utterback T.,Aken S.E.V.,Feldblyum T.V.D'Ascenzo M.,Deng W.L.,Ramos A.R.,Alfano J.R.,Cartinhour S.,Chatterjee A.K.,Delaney T.P.,Lazarowitz S.G.Martin G.B.,Schneider D.J.,Tang X.Y.,Bender C.L.,White O.,Fraser C.M.,and Collmer A.,2003,The complete genome sequence of the Arabidopsis and tomato pathogen Pseudomonas syringae pv.tomato DC3000,Proc.Natl.Acad Sci.USA,100(18):10181-10186
Dormann P.,Gopalan S.,He S.Y.,and Benning C.,2000,A gene family in Arabidopsis thaliana with sequence similarity to NDR1 and HIN1,Plant Physiology and Biochemistry,38(10):789-796
Edwards T.A.,Wilkinson B.D.,Wharton R.B.,and Aggarwal AK.,2003,Model of the Brain Tumor-Pumilio translation repressor complex,Genes Dev.,17(20):2508-2513
Felix G.,Duran J.D.,Volko S.,and Boller T.,1999,Plants have a sensitive perception system for the most conserved domain of bacterial flagellin,Plant J.,18(3):265-276
Fridell R.A.,Harding L.S.,Bogerd H.P.,and Cullen B.R.,1995Identification of a novel human zinc finger protein tha specifically interacts with the activation domain of lentiviral Tat proteins,Virology.,209(2):347-357
Gu Y.X.,and Wang D.J.,2003,Effect of external salicylic acid on Curvularia lunate boed on Festuca arundinacea schreb Zhongguo Caodi(Grassland of China),25(4):56-60,71(古燕翔,王代军,2003,外源诱导物水杨酸对草坪型高羊茅弯孢霉叶斑病抗性影响的研究,中国草地,25(4):56-60,71)
Hao C.C.,Yang P.,Chen Z.,Jia J.,Zhao B.,Si H.L.,Han J.M.,Xing J.H.,and Dong J.H.,2013,Mechanism analysis of At BT4 from Arabidopsis thaliana against Botrytis cinerea,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),46(9):1756-1761(郝丛丛,杨萍,陈展,贾娇,赵斌,司贺龙,韩建民,邢继红,董金皋,2013,拟南芥At BT4对灰葡萄孢的抗性机制分析,中国农业科学,46(9):1756-1761)
Hong Y.M.,and Zhao T.C.,2006,Advanced in the signal research of Pto-mediated resistance to bacterial dpeck disease in tomato,Zhongguo Nongxue Tongbao(Chinese Agricultural Science Bulletin),22(1):259-263(洪玉梅,赵廷昌,2006,番茄细菌性斑点病抗病信号传导途径的研究进展,中国农学通报,22(1):259-263)
Hu B.,Jin J.,Guo A.Y.,Zhang H.,Luo J.,and Gao G.,2015,GSDS 2.0:an upgraded gene feature visualization server,Bioinformatics,31(8):1296-1297
Kumar S.,Stecher G.,and Tamura K.,2016,MEGA7:Molecular evolutionary genetics analysis version 7.0 for bigger datasets,Mol.Biol.Evol.,33(7):1870-1874
Larkin M.A.,Blackshields G.,Brown N.P.,Chenna R.,McGettigan P.A.,McWilliam H.,Valentin F.,Wallace I.M.,Wilm A.,Lopez R.,Thompson J.D.,Gibson T.J.,and Higgins D.G.,2007,Clustal W and Clustal X version 2.0,Bioinformatics,23(21):2947-2948
Li S.J.,Zhu T.H.,Huang Y.N.,Han S.,and Qiao T.M.,2012,Roles of salicylic acid in inducing resistance of Camellia to the gray spot and the biochemical responses,Linye Kexue(Scientia Silvae Sinicae),48(2):103-109(李姝江,朱天辉,黄艳娜,韩珊,谯天敏,2012,水杨酸诱导山茶抗灰斑病的作用及生理生化响应,林业科学,48(2):103-109)
Li T.T.,Gao Y.F.,Ma X.,Chen Y.F.,Wang Y.,and Ma J.B.,2016,Effects of exogenous brassinosteroid on photosynthesis of three spesies of Populus under drought,salt and copper stress,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),36(1):218-226(李涛涛,高永峰,马瑄,陈永富,王阳,马金彪,2016,外源油菜素内酯对三种杨树在干旱、盐和铜胁迫下光合生理的影响,基因组学与应用生物学,36(1):218-226)
Li Y.,Wang F.,Tan G.F.,Jia X.L.,Jiang Q.,and Xiong A.S.,2014,Cloning and expression pattern analysis of NHL-like protein gene in celery,Zhiwu Yichaun Ziyaun Xuebao(Journal of Plant Genetic Resources),15(4):788-794(李岩,王枫,谭国飞,贾晓玲,蒋倩,熊爱生,2014,芹菜NHL-like蛋白基因克隆与表达分析,植物遗传资源学报,15(4):788-794)
Liu Z.L.,Zhang S.L.,and Gao F.Y.,2011,Effect of exogenous salicylic acid on antioxidant enzymes and Ca2+density in pear leaves infected by Physalosproa piricola nose,Yingyong Yu Huanjingshengwu Xuebao(Chinese Journal of Applied and Environmental Biology),17(2):215-218(刘招龙张绍铃,高富永,2011,外源水杨酸对梨叶片感染轮纹病菌后抗氧化酶活性及Ca2+浓度的影响,应用与环境生物学报,17(2):215-218)
Loedige I.,Gaidatzis D.,Sack R.,Meister G.,and Filipowicz W.2013,The mammalian TRIM-NHL protein TRIM71/LIN-41is a repressor of m RNA function,Nucleic Acids Res.,41(1)518-532
Loedige I.,Stotz M.,Qamar S.,Kramer K.,Hennig J.,Schubert T.,Loffler P.,Langst G.,Merkl R.,Urlaub H.,and Meister G.,2014,The NHL domain of BRAT is an RNA-binding domain that directly contacts the hunchback m RNA for regulation,Genes Dev.,28(7):749-764
Mao A.J.,Wang Y.J.,Feng L.X.,Geng S.X.,and Xu Y.,2004Study on the resistance induced by salicylic acid against Phytophthora capsici in pepper,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),37(10):1481-1486(毛爱军王永健,冯兰香,耿三省,许勇,2004,水杨酸等4种诱导剂诱导辣椒抗疫病作用的研究,中国农业科学,37(10):1481-1486)
Slack F.,and Ruvkun G.,1997,Temporal pattern formation by heterochronic genes,Annu.Rev.Genet.,31:11-34
Slack F.J.,and Ruvkun G.,1998,A novel repeat domain that is often associated with RING finger and B-box motifs,Trends Biochem.Sci.,23(12):474-475
Sonoda J.,and Wharton R.P.,2001,Drosophila brain tumor is a translational repressor,Genes Dev.,15(6):762-773
Tsuda K.,Sato M.,Glazebrook J.,Cohen J.D.,and Katagiri F.,2008,Interplay between MAMP-triggered and SA-mediated defense responses,Plant J.,53(5):763-775
Xu M.,Sun Z.M.,Liu S.A.,Chen C.H.,Xu L.A.,and Huang M.R.,2016,Cloning and expression analysis of salinity stress related PeuHKT1 gene from Populus euphratica,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(9):2312-2318(胥猛,孙子谋,刘思安,陈彩慧,徐立安,黄敏仁,2016,胡杨耐盐基因PeuHKT1的克隆与表达分析,分子植物育种,14(9):2312-2318)
Xu M.,Zhang B.,Su X.,Zhang S.,and Huang M.,2011,Reference gene selection for quantitative real-time polymerase chain reaction in Populus,Anal.Biochem.,408(2):337-339
Zhao H.,Wang S.Q.,Yu X.Y.,Qiang C.K.,and Chen C.J.,2016,Analysis of the resistance of Botrytis Cinerea from strawberry in Nanjing and Zhenjiang area to six fungicides and its biological characteristics,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(7):1828-1834(赵虎,王松群,余新燕,强承魁,陈长军,2016,南京,镇江地区草莓灰霉病菌对6种杀菌剂的抗药性及生物学性状分析,基因组学与应用生物学,35(7):1828-1834)
Bari R.,and Jones J.D.,2009,Role of plant hormones in plan defence responses,Plant Mol.Biol.,69(4):473-488
Buell C.R.,Joardar V.,Lindeberg M.,Selengut J.,Paulsen I.T.Gwinn M.L.,Dodson R.J.,Deboy R.T.,Durkin A.S.,Kolonay J.F.,Madupu R.,Daugherty S.,Brinkac L.,Beanan M.J.Haft D.H.,Nelson W.C.,Davidsen T.,Zafar N.,Zhou L.W.Liu J.,Yuan Q.P.,Khouri H.,Fedorova N.,Tran B.,Russel D.,Berry K.,Utterback T.,Aken S.E.V.,Feldblyum T.V.D'Ascenzo M.,Deng W.L.,Ramos A.R.,Alfano J.R.,Cartinhour S.,Chatterjee A.K.,Delaney T.P.,Lazarowitz S.G.Martin G.B.,Schneider D.J.,Tang X.Y.,Bender C.L.,White O.,Fraser C.M.,and Collmer A.,2003,The complete genome sequence of the Arabidopsis and tomato pathogen Pseudomonas syringae pv.tomato DC3000,Proc.Natl.Acad Sci.USA,100(18):10181-10186
Dormann P.,Gopalan S.,He S.Y.,and Benning C.,2000,A gene family in Arabidopsis thaliana with sequence similarity to NDR1 and HIN1,Plant Physiology and Biochemistry,38(10):789-796
Edwards T.A.,Wilkinson B.D.,Wharton R.B.,and Aggarwal AK.,2003,Model of the Brain Tumor-Pumilio translation repressor complex,Genes Dev.,17(20):2508-2513
Felix G.,Duran J.D.,Volko S.,and Boller T.,1999,Plants have a sensitive perception system for the most conserved domain of bacterial flagellin,Plant J.,18(3):265-276
Fridell R.A.,Harding L.S.,Bogerd H.P.,and Cullen B.R.,1995Identification of a novel human zinc finger protein tha specifically interacts with the activation domain of lentiviral Tat proteins,Virology.,209(2):347-357
Gu Y.X.,and Wang D.J.,2003,Effect of external salicylic acid on Curvularia lunate boed on Festuca arundinacea schreb Zhongguo Caodi(Grassland of China),25(4):56-60,71(古燕翔,王代军,2003,外源诱导物水杨酸对草坪型高羊茅弯孢霉叶斑病抗性影响的研究,中国草地,25(4):56-60,71)
Hao C.C.,Yang P.,Chen Z.,Jia J.,Zhao B.,Si H.L.,Han J.M.,Xing J.H.,and Dong J.H.,2013,Mechanism analysis of At BT4 from Arabidopsis thaliana against Botrytis cinerea,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),46(9):1756-1761(郝丛丛,杨萍,陈展,贾娇,赵斌,司贺龙,韩建民,邢继红,董金皋,2013,拟南芥At BT4对灰葡萄孢的抗性机制分析,中国农业科学,46(9):1756-1761)
Hong Y.M.,and Zhao T.C.,2006,Advanced in the signal research of Pto-mediated resistance to bacterial dpeck disease in tomato,Zhongguo Nongxue Tongbao(Chinese Agricultural Science Bulletin),22(1):259-263(洪玉梅,赵廷昌,2006,番茄细菌性斑点病抗病信号传导途径的研究进展,中国农学通报,22(1):259-263)
Hu B.,Jin J.,Guo A.Y.,Zhang H.,Luo J.,and Gao G.,2015,GSDS 2.0:an upgraded gene feature visualization server,Bioinformatics,31(8):1296-1297
Kumar S.,Stecher G.,and Tamura K.,2016,MEGA7:Molecular evolutionary genetics analysis version 7.0 for bigger datasets,Mol.Biol.Evol.,33(7):1870-1874
Larkin M.A.,Blackshields G.,Brown N.P.,Chenna R.,McGettigan P.A.,McWilliam H.,Valentin F.,Wallace I.M.,Wilm A.,Lopez R.,Thompson J.D.,Gibson T.J.,and Higgins D.G.,2007,Clustal W and Clustal X version 2.0,Bioinformatics,23(21):2947-2948
Li S.J.,Zhu T.H.,Huang Y.N.,Han S.,and Qiao T.M.,2012,Roles of salicylic acid in inducing resistance of Camellia to the gray spot and the biochemical responses,Linye Kexue(Scientia Silvae Sinicae),48(2):103-109(李姝江,朱天辉,黄艳娜,韩珊,谯天敏,2012,水杨酸诱导山茶抗灰斑病的作用及生理生化响应,林业科学,48(2):103-109)
Li T.T.,Gao Y.F.,Ma X.,Chen Y.F.,Wang Y.,and Ma J.B.,2016,Effects of exogenous brassinosteroid on photosynthesis of three spesies of Populus under drought,salt and copper stress,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),36(1):218-226(李涛涛,高永峰,马瑄,陈永富,王阳,马金彪,2016,外源油菜素内酯对三种杨树在干旱、盐和铜胁迫下光合生理的影响,基因组学与应用生物学,36(1):218-226)
Li Y.,Wang F.,Tan G.F.,Jia X.L.,Jiang Q.,and Xiong A.S.,2014,Cloning and expression pattern analysis of NHL-like protein gene in celery,Zhiwu Yichaun Ziyaun Xuebao(Journal of Plant Genetic Resources),15(4):788-794(李岩,王枫,谭国飞,贾晓玲,蒋倩,熊爱生,2014,芹菜NHL-like蛋白基因克隆与表达分析,植物遗传资源学报,15(4):788-794)
Liu Z.L.,Zhang S.L.,and Gao F.Y.,2011,Effect of exogenous salicylic acid on antioxidant enzymes and Ca2+density in pear leaves infected by Physalosproa piricola nose,Yingyong Yu Huanjingshengwu Xuebao(Chinese Journal of Applied and Environmental Biology),17(2):215-218(刘招龙张绍铃,高富永,2011,外源水杨酸对梨叶片感染轮纹病菌后抗氧化酶活性及Ca2+浓度的影响,应用与环境生物学报,17(2):215-218)
Loedige I.,Gaidatzis D.,Sack R.,Meister G.,and Filipowicz W.2013,The mammalian TRIM-NHL protein TRIM71/LIN-41is a repressor of m RNA function,Nucleic Acids Res.,41(1)518-532
Loedige I.,Stotz M.,Qamar S.,Kramer K.,Hennig J.,Schubert T.,Loffler P.,Langst G.,Merkl R.,Urlaub H.,and Meister G.,2014,The NHL domain of BRAT is an RNA-binding domain that directly contacts the hunchback m RNA for regulation,Genes Dev.,28(7):749-764
Mao A.J.,Wang Y.J.,Feng L.X.,Geng S.X.,and Xu Y.,2004Study on the resistance induced by salicylic acid against Phytophthora capsici in pepper,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),37(10):1481-1486(毛爱军王永健,冯兰香,耿三省,许勇,2004,水杨酸等4种诱导剂诱导辣椒抗疫病作用的研究,中国农业科学,37(10):1481-1486)
Slack F.,and Ruvkun G.,1997,Temporal pattern formation by heterochronic genes,Annu.Rev.Genet.,31:11-34
Slack F.J.,and Ruvkun G.,1998,A novel repeat domain that is often associated with RING finger and B-box motifs,Trends Biochem.Sci.,23(12):474-475
Sonoda J.,and Wharton R.P.,2001,Drosophila brain tumor is a translational repressor,Genes Dev.,15(6):762-773
Tsuda K.,Sato M.,Glazebrook J.,Cohen J.D.,and Katagiri F.,2008,Interplay between MAMP-triggered and SA-mediated defense responses,Plant J.,53(5):763-775
Xu M.,Sun Z.M.,Liu S.A.,Chen C.H.,Xu L.A.,and Huang M.R.,2016,Cloning and expression analysis of salinity stress related PeuHKT1 gene from Populus euphratica,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(9):2312-2318(胥猛,孙子谋,刘思安,陈彩慧,徐立安,黄敏仁,2016,胡杨耐盐基因PeuHKT1的克隆与表达分析,分子植物育种,14(9):2312-2318)
Xu M.,Zhang B.,Su X.,Zhang S.,and Huang M.,2011,Reference gene selection for quantitative real-time polymerase chain reaction in Populus,Anal.Biochem.,408(2):337-339
Zhao H.,Wang S.Q.,Yu X.Y.,Qiang C.K.,and Chen C.J.,2016,Analysis of the resistance of Botrytis Cinerea from strawberry in Nanjing and Zhenjiang area to six fungicides and its biological characteristics,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(7):1828-1834(赵虎,王松群,余新燕,强承魁,陈长军,2016,南京,镇江地区草莓灰霉病菌对6种杀菌剂的抗药性及生物学性状分析,基因组学与应用生物学,35(7):1828-1834)
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