花生AhLRPK1基因的克隆及生物信息学分析
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摘要
植物受体蛋白激酶参与植物的生长发育,抗逆抗病防御反应、细胞分化、在宿主与病原菌互作过程中起着重要的作用。目前在花生上蛋白激酶基因研究较少。本研究基于前期多态性SNP标记的开发和QTL定位,获得了一个抗青枯病候选蛋白激酶基因;通过RT-PCR克隆技术获得了一段长为2 154 bp的ORF序列,暂命名为AhLRPK1;采用生物信息学方法预测并分析AhLRPK1基因编码的蛋白质常规理化性质,跨膜结构域、进化分析和高级结构等。结果表明AhLRPK1基因编码717个氨基酸为稳定的亲水性蛋白,含有一个信号肽、跨膜结构域、多个LRR基序以及一个激酶结构域;与拟南芥LRR亚家族进化同源性分析显示AhLRPK1属于LRRⅤ亚家族,与拟南芥的AT3G13065蛋白亲缘关系最近;AhLRPK1基因在花生基因芯片中表达模式分析表明,在茎和花中的表达量最大,在青枯菌诱导情况下,AhLRPK1基因在抗感花生品种中都表现出下调表达的趋势,AhLRPK1基因受乙烯利表达下调。这些结果有助于进一步验证其生物功能。
Receptor protein kinase is involved in plant growth and development, stress and defense response, cell differentiation, and plays an important role in the interaction between host and pathogen. At present, there are few studies on protein kinase genes in peanut. In this study, a bacterial wilt resistance-related candidate receptor-like kinase gene was obtained by SNP polymorphism markers development and QTL mapping, named AhLPRK1. The ORF sequence was cloned by RT-PCR from resistant peanut cultivar Yueyou 92 containing 2 154 bp which was encoded 717 amino acid. The physicochemical properties transmembrane domain, evolutionary analysis and advanced structure were predicted and analyzed by bioinformatics methods. These predictions were shown conserved AhLPRK1 was a stable hydrophilic protein, included a signal peptide, transmembrane helices domains, multiple LRR motif and a kinase domain structure. Phylogenetic tree analysis showed AhLRPK1 belonged to LRRV subfamily, and was closed to AT3 G13065. In the peanut microarray, the expression pattern of AhLRPK1 showed higher in stem and flower than other organs, down-regulation challenged by Rastonia solanacearum in the resistant peanut cultivar as well as in the susceptible cultivar. The expression of AhLRPK1 was down regulated by ethephon.These results lay the foundation for further verify its biological function.
Receptor protein kinase is involved in plant growth and development, stress and defense response, cell differentiation, and plays an important role in the interaction between host and pathogen. At present, there are few studies on protein kinase genes in peanut. In this study, a bacterial wilt resistance-related candidate receptor-like kinase gene was obtained by SNP polymorphism markers development and QTL mapping, named AhLPRK1. The ORF sequence was cloned by RT-PCR from resistant peanut cultivar Yueyou 92 containing 2 154 bp which was encoded 717 amino acid. The physicochemical properties transmembrane domain, evolutionary analysis and advanced structure were predicted and analyzed by bioinformatics methods. These predictions were shown conserved AhLPRK1 was a stable hydrophilic protein, included a signal peptide, transmembrane helices domains, multiple LRR motif and a kinase domain structure. Phylogenetic tree analysis showed AhLRPK1 belonged to LRRV subfamily, and was closed to AT3 G13065. In the peanut microarray, the expression pattern of AhLRPK1 showed higher in stem and flower than other organs, down-regulation challenged by Rastonia solanacearum in the resistant peanut cultivar as well as in the susceptible cultivar. The expression of AhLRPK1 was down regulated by ethephon.These results lay the foundation for further verify its biological function.
引文
Antolín-Llovera M.,Ried M.K.,Binder A.,and Parniske M.,2012,Receptor kinase signaling pathways in plant-microbe interactions,Annu.Rev.Phytopathol.,50(50):451-473
Chen Y.L.,2016,Analysis of disease-resistance pathway of the receptor like kinase St LRPK1,Thesis for M.S.,College of Horticulture and Forestry,Huazhong Agricultural University,Supervisor:Tian Z.D.,pp.35-38(陈艳林,2016,类受体激酶St LRPK1参与的抗病信号途经分析,硕士学位论文,华中农业大学园艺林学学院,导师:田振东,pp.35-38)
Clark S.E.,Running M.P.,and Meyerowitz E.M.,1993,CLAVATA1,a regulator of meristem and flower development in Arabidopsis,Development,119(2):397-418
Duan X.H.,Geng L.L.,Shu Z.L.,Zhu Y.M.,and Zhang J.,2012,Cloning and bioinfomatic analysis of symrk gene cloned from Arachis hypogaea L.,Zhongguo Nongye Keji Daobao(Journal of Agricultural Science and Technology),14(5):33-41(段小红,耿丽丽,束长龙,朱延明,张杰,2012,花生共生受体类蛋白激酶基因的克隆及生物信息学分析,中国农业科技导报,14(5):33-41)
Eyüboglu B.,Pfister K.,Haberer G.,Chevalier D.,Fuchs A.,Mayer K.F.,and Schneitz K.,2007,Molecular characterisation of the STRUBBELIG-RECEPTOR FAMILY of genes encoding putative leucine-rich repeat receptor-like kinases in Arabidopsis thaliana,BMC Plant Biology,7(1):16
Jun Z.,Zhang Z.,Gao Y.,Zhou L.,Fang L.,Chen X.,Ning Z.,Chen T.,Guo W.,and Zhang T.,2015,Overexpression of Gb RLK,a putative receptor-like kinase gene,improved cotton tolerance to Verticillium wilt,Scientific Reports,5:15048
Lehti-Shiu M.D.,and Shiu S.H.,2012,Diversity,classification and function of the plant protein kinase superfamily,Philos.Trans.R.Soc.Lond.B Biol.Sci.,367(1602):2619-2639
Feng L.,Zhang H.W.,and Huang R.F.,2012,Research progress on LRR receptor-like protein kinase in plant,Zhongguo Nongye Keji Daobao(Journal of Agricultural Science and T-echnology),14(6):43-48(冯蕾,张海文,黄荣峰,2012,植物LRR类受体蛋白激酶的研究进展,中国农业科技导报,14(6):43-48)
Li Y.D.,and He J.G.,2011,The diseases resistance analysis of Os BRR1,A putative rice receptor like kinase,Huabeinong Xuebao(Acta Agriculturae Boreali-Sinica),26(4):27-31(李亚栋,何近刚,2011,水稻类受体激酶Os BRR1对稻瘟病抗性分析,华北农学报,26(4):27-31)
Pei L.L.,Guo Y.H.,Xu Z.S.,Li L.C.,Chen M.,and Ma Y.Z.2012,Research progress on stress-related protein kinases in plants,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),32(5):1052-1061(裴丽丽,郭玉华,徐兆师李连城,陈明,马有志,2012,植物逆境胁迫相关蛋白激酶的研究进展,西北植物学报,32(5):1052-1061)
Mizuno S.,Osakabe Y.,Maruyama K.,Ito T.,Osakabe K.,Sato T.,Shinozaki K.,and Yamaguchi-Shinozaki K.,2007,Receptorlike protein kinase 2(RPK 2)is a novel factor controlling anther development in Arabidopsis thaliana,Plant J.,50(5)751-766
Ouyang S.Q.,Liu Y.F.,Liu P.,Lei G.,He S.J.,Ma B.,Zhang WK.,Zhang J.S.,and Chen S.Y.,2010,Receptor-like kinase Os SIK1 improves drought and salt stress tolerance in rice(Oryza sativa)plants,Plant J.,62(2):316-329
Shiu S.H.,Karlowski W.M.,Pan R.,Tzeng Y.H.,Mayer K.F.and Li W.H.,2004,Comparative analysis of the receptorlike kinase family in Arabidopsis and rice,Plant Cell,16(5)1220-1234
Sun X.L.,Yu Q.Y.,Tang L.L.,Ji W.,Bai X.,Cai H.,Liu X.F.Ding X.D.,and Zhu Y.M.,2013,Gs SRK,a G-type lectin S-receptor-like serine/threonine protein kinase,is a positive regulator of plant tolerance to salt stress,J.Plant Physiol.170(5):505-515
Tan H.,2015,Function analysis of receptor-like kinase RLK80 in Arabidopsis thaliana,Thesis for M.S,Hunan Normal University,Supervisor:Li D.P.,and Tian L.F.,pp.41-44(谭虹2015,拟南芥类受体蛋白激酶RLK80的功能研究,硕士学位论文,湖南师范大学,导师:李东屏,田连福,pp41-44)
Walker J.C.,and Zhang R.,1990,Relationship of a putative receptor protein kinase from maize to the S-locus glycoproteins of Brassica,Nature,345(6277):743-746
Wang J.,Zhang P.,Liu S.,Cong B.,and Chen K.,2016,A leucine-rich repeat receptor-like kinase from the antarctic moss Pohlia nutans confers salinity and ABA stress tolerance Plant Molecular Biology Reporter,34(6):1136-1145
Wu P.C.,2008,Screening of pathogenesis related proteins mediated by rice disease resistance gene Xa21,Thesis for M.SAgricultural University of Hebei,Supervisor:Liu Z.G.,pp30-34(武鹏程,2008,水稻Xa21抗病相关蛋白质的筛选硕士学位论文,河北农业大学,导师:刘振国,pp.30-34)
Wu T.,Tian Z.,Liu J.,and Xie C.,2009,A novel leucine-rich repeat receptor-like kinase gene in potato,St LRPK1,is involved in response to diverse stresses,Mol.Biol.Rep.,36(8):2365-2374
Wu Y.,Xun Q.,Guo Y.,Zhang J.,Cheng K.,Shi T.,He K.,Hou S.,Gou X.,and Li J.,2016,Genome-wide expression pattern analyses of the Arabidopsis leucine-rich repeat receptor-like kinases,Mol.Plant,9(2):289-300
Xiong F.Q.,Liu J.X.,Wang C.C.,He L.Q.,Han Z.Q.,Jiang J.,Zhong R.C.,Li Z.,Tang X.M.,and Tang R.H.,2013,m CTA-B-d Li Cl method for efficiently extracting RNA from various tissues of cultivated peanuts and its vertification,Nanfang Nongye Xuebao(Journal of Southern Agricultura),44(11):1781-1784(熊发前,刘俊仙,王丛丛,贺梁琼,韩柱强,蒋菁,钟瑞春,李忠,唐秀梅,唐荣华,2013,m CTAB-d Li Cl法高效提取花生各组织部位RNA及其验证,南方农业学报,44(11):1781-1784)
Yadav R.K.,Fulton L.,Batoux M.,and Schneitz K.,2008,The Arabidopsis receptor-like kinase STRUBBELIG mediates inter-cell-layer signaling during floral development,Dev.Biol.,323(2):261-270
Yan H.Y.,Zong C.Z.,Jing C.H.,and Dan S.H.,2011,Analysis of relativity of receptor-like kinase to Aspergillus flavus resistance in peanut,Huabeinong Xuebao(Acta Agriculturae Boreali-Sinica),26(4):198-201(严海燕,宗成志,景聪慧,单世华,2011,花生黄曲霉抗性与类受体蛋白激酶的相关性分析,华北农学报,26(4):198-201)
Yang L.,Wu K.C.,Gao P.,Liu X.J.,Li G.P.,and Wu Z.J.,2014,Gs LRPK,a novel cold-activated leucine-rich repeat receptor-like protein kinase from Glycine soja,is a positive regulator to cold stress tolerance,Plant Sci.,215-216:19-28
Yang X.,Deng F.,and Ramonell K.M.,2012,Recep tor-like kinases and receptor-like proteins:keys to pathogen recognition and defense signaling in plant innate immunity,Frontiers in Biology,7(2):155-166
Yin M.,Wang Y.,Zhang L.,Li J.,Quan W.,Yang L.,Wang Q.,and Chan Z.,2017,The Arabidopsis Cys2/His2 zinc finger transcription factor ZAT18 is a positive regulator of plant tolerance to drought stress,J.Exp.Bot.,68(11):2991-3005
Zhang C.,2013,Preliminary analysis of resistance molecu mechanism to bacterial wilt in peanut,Dissertation for Ph.D.,College of Life Science,Fujian Agriculture and Forestry University,Supervisor:Zhuang W.J.,pp.94-96(张冲,2013,花生抗青枯病分子机理初步解析,博士学位论文,福建农林大学生命科学学院,导师:庄伟建,pp.94-96)
Zhao J.,Gao Y.,Zhang Z.,Chen T.,Guo W.,and Zhang T.,2013,A receptor-like kinase gene(Gb RLK)from Gossypium barbadense enhances salinity and drought-stress tolerance in Arabidopsis,BMC Plant Biol.,13(1):110
Zhou Q.H.,Li C.Q.,and Kuang Q.,2003,Advancements in plant protein kinase,Shengwuxue Zazhi(Journal of Biology),20(3):1-4(周庆红,李成琼,匡全,2003,植物蛋白激酶研究进展,生物学杂志,20(3):1-4)
Zhu Y.,Peng Z.Y.,Fan Z.X.,Zhang B.,Wang C.X.,Zheng L.,and Bi Y.P.,2013,Cloning and sequence analysis of protein kinase gene Ah STK from Arachis hypogaea L.,Shandong Nongye Kexue(Shandong Agricultural Sciences)45(1):9-13(朱岩,彭振英,范仲学,张斌,王成祥,郑玲,毕玉平,2013,花生蛋白激酶基因Ah STK的克隆与序列分析,山东农业科学,45(1):9-13)
Chen Y.L.,2016,Analysis of disease-resistance pathway of the receptor like kinase St LRPK1,Thesis for M.S.,College of Horticulture and Forestry,Huazhong Agricultural University,Supervisor:Tian Z.D.,pp.35-38(陈艳林,2016,类受体激酶St LRPK1参与的抗病信号途经分析,硕士学位论文,华中农业大学园艺林学学院,导师:田振东,pp.35-38)
Clark S.E.,Running M.P.,and Meyerowitz E.M.,1993,CLAVATA1,a regulator of meristem and flower development in Arabidopsis,Development,119(2):397-418
Duan X.H.,Geng L.L.,Shu Z.L.,Zhu Y.M.,and Zhang J.,2012,Cloning and bioinfomatic analysis of symrk gene cloned from Arachis hypogaea L.,Zhongguo Nongye Keji Daobao(Journal of Agricultural Science and Technology),14(5):33-41(段小红,耿丽丽,束长龙,朱延明,张杰,2012,花生共生受体类蛋白激酶基因的克隆及生物信息学分析,中国农业科技导报,14(5):33-41)
Eyüboglu B.,Pfister K.,Haberer G.,Chevalier D.,Fuchs A.,Mayer K.F.,and Schneitz K.,2007,Molecular characterisation of the STRUBBELIG-RECEPTOR FAMILY of genes encoding putative leucine-rich repeat receptor-like kinases in Arabidopsis thaliana,BMC Plant Biology,7(1):16
Jun Z.,Zhang Z.,Gao Y.,Zhou L.,Fang L.,Chen X.,Ning Z.,Chen T.,Guo W.,and Zhang T.,2015,Overexpression of Gb RLK,a putative receptor-like kinase gene,improved cotton tolerance to Verticillium wilt,Scientific Reports,5:15048
Lehti-Shiu M.D.,and Shiu S.H.,2012,Diversity,classification and function of the plant protein kinase superfamily,Philos.Trans.R.Soc.Lond.B Biol.Sci.,367(1602):2619-2639
Feng L.,Zhang H.W.,and Huang R.F.,2012,Research progress on LRR receptor-like protein kinase in plant,Zhongguo Nongye Keji Daobao(Journal of Agricultural Science and T-echnology),14(6):43-48(冯蕾,张海文,黄荣峰,2012,植物LRR类受体蛋白激酶的研究进展,中国农业科技导报,14(6):43-48)
Li Y.D.,and He J.G.,2011,The diseases resistance analysis of Os BRR1,A putative rice receptor like kinase,Huabeinong Xuebao(Acta Agriculturae Boreali-Sinica),26(4):27-31(李亚栋,何近刚,2011,水稻类受体激酶Os BRR1对稻瘟病抗性分析,华北农学报,26(4):27-31)
Pei L.L.,Guo Y.H.,Xu Z.S.,Li L.C.,Chen M.,and Ma Y.Z.2012,Research progress on stress-related protein kinases in plants,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),32(5):1052-1061(裴丽丽,郭玉华,徐兆师李连城,陈明,马有志,2012,植物逆境胁迫相关蛋白激酶的研究进展,西北植物学报,32(5):1052-1061)
Mizuno S.,Osakabe Y.,Maruyama K.,Ito T.,Osakabe K.,Sato T.,Shinozaki K.,and Yamaguchi-Shinozaki K.,2007,Receptorlike protein kinase 2(RPK 2)is a novel factor controlling anther development in Arabidopsis thaliana,Plant J.,50(5)751-766
Ouyang S.Q.,Liu Y.F.,Liu P.,Lei G.,He S.J.,Ma B.,Zhang WK.,Zhang J.S.,and Chen S.Y.,2010,Receptor-like kinase Os SIK1 improves drought and salt stress tolerance in rice(Oryza sativa)plants,Plant J.,62(2):316-329
Shiu S.H.,Karlowski W.M.,Pan R.,Tzeng Y.H.,Mayer K.F.and Li W.H.,2004,Comparative analysis of the receptorlike kinase family in Arabidopsis and rice,Plant Cell,16(5)1220-1234
Sun X.L.,Yu Q.Y.,Tang L.L.,Ji W.,Bai X.,Cai H.,Liu X.F.Ding X.D.,and Zhu Y.M.,2013,Gs SRK,a G-type lectin S-receptor-like serine/threonine protein kinase,is a positive regulator of plant tolerance to salt stress,J.Plant Physiol.170(5):505-515
Tan H.,2015,Function analysis of receptor-like kinase RLK80 in Arabidopsis thaliana,Thesis for M.S,Hunan Normal University,Supervisor:Li D.P.,and Tian L.F.,pp.41-44(谭虹2015,拟南芥类受体蛋白激酶RLK80的功能研究,硕士学位论文,湖南师范大学,导师:李东屏,田连福,pp41-44)
Walker J.C.,and Zhang R.,1990,Relationship of a putative receptor protein kinase from maize to the S-locus glycoproteins of Brassica,Nature,345(6277):743-746
Wang J.,Zhang P.,Liu S.,Cong B.,and Chen K.,2016,A leucine-rich repeat receptor-like kinase from the antarctic moss Pohlia nutans confers salinity and ABA stress tolerance Plant Molecular Biology Reporter,34(6):1136-1145
Wu P.C.,2008,Screening of pathogenesis related proteins mediated by rice disease resistance gene Xa21,Thesis for M.SAgricultural University of Hebei,Supervisor:Liu Z.G.,pp30-34(武鹏程,2008,水稻Xa21抗病相关蛋白质的筛选硕士学位论文,河北农业大学,导师:刘振国,pp.30-34)
Wu T.,Tian Z.,Liu J.,and Xie C.,2009,A novel leucine-rich repeat receptor-like kinase gene in potato,St LRPK1,is involved in response to diverse stresses,Mol.Biol.Rep.,36(8):2365-2374
Wu Y.,Xun Q.,Guo Y.,Zhang J.,Cheng K.,Shi T.,He K.,Hou S.,Gou X.,and Li J.,2016,Genome-wide expression pattern analyses of the Arabidopsis leucine-rich repeat receptor-like kinases,Mol.Plant,9(2):289-300
Xiong F.Q.,Liu J.X.,Wang C.C.,He L.Q.,Han Z.Q.,Jiang J.,Zhong R.C.,Li Z.,Tang X.M.,and Tang R.H.,2013,m CTA-B-d Li Cl method for efficiently extracting RNA from various tissues of cultivated peanuts and its vertification,Nanfang Nongye Xuebao(Journal of Southern Agricultura),44(11):1781-1784(熊发前,刘俊仙,王丛丛,贺梁琼,韩柱强,蒋菁,钟瑞春,李忠,唐秀梅,唐荣华,2013,m CTAB-d Li Cl法高效提取花生各组织部位RNA及其验证,南方农业学报,44(11):1781-1784)
Yadav R.K.,Fulton L.,Batoux M.,and Schneitz K.,2008,The Arabidopsis receptor-like kinase STRUBBELIG mediates inter-cell-layer signaling during floral development,Dev.Biol.,323(2):261-270
Yan H.Y.,Zong C.Z.,Jing C.H.,and Dan S.H.,2011,Analysis of relativity of receptor-like kinase to Aspergillus flavus resistance in peanut,Huabeinong Xuebao(Acta Agriculturae Boreali-Sinica),26(4):198-201(严海燕,宗成志,景聪慧,单世华,2011,花生黄曲霉抗性与类受体蛋白激酶的相关性分析,华北农学报,26(4):198-201)
Yang L.,Wu K.C.,Gao P.,Liu X.J.,Li G.P.,and Wu Z.J.,2014,Gs LRPK,a novel cold-activated leucine-rich repeat receptor-like protein kinase from Glycine soja,is a positive regulator to cold stress tolerance,Plant Sci.,215-216:19-28
Yang X.,Deng F.,and Ramonell K.M.,2012,Recep tor-like kinases and receptor-like proteins:keys to pathogen recognition and defense signaling in plant innate immunity,Frontiers in Biology,7(2):155-166
Yin M.,Wang Y.,Zhang L.,Li J.,Quan W.,Yang L.,Wang Q.,and Chan Z.,2017,The Arabidopsis Cys2/His2 zinc finger transcription factor ZAT18 is a positive regulator of plant tolerance to drought stress,J.Exp.Bot.,68(11):2991-3005
Zhang C.,2013,Preliminary analysis of resistance molecu mechanism to bacterial wilt in peanut,Dissertation for Ph.D.,College of Life Science,Fujian Agriculture and Forestry University,Supervisor:Zhuang W.J.,pp.94-96(张冲,2013,花生抗青枯病分子机理初步解析,博士学位论文,福建农林大学生命科学学院,导师:庄伟建,pp.94-96)
Zhao J.,Gao Y.,Zhang Z.,Chen T.,Guo W.,and Zhang T.,2013,A receptor-like kinase gene(Gb RLK)from Gossypium barbadense enhances salinity and drought-stress tolerance in Arabidopsis,BMC Plant Biol.,13(1):110
Zhou Q.H.,Li C.Q.,and Kuang Q.,2003,Advancements in plant protein kinase,Shengwuxue Zazhi(Journal of Biology),20(3):1-4(周庆红,李成琼,匡全,2003,植物蛋白激酶研究进展,生物学杂志,20(3):1-4)
Zhu Y.,Peng Z.Y.,Fan Z.X.,Zhang B.,Wang C.X.,Zheng L.,and Bi Y.P.,2013,Cloning and sequence analysis of protein kinase gene Ah STK from Arachis hypogaea L.,Shandong Nongye Kexue(Shandong Agricultural Sciences)45(1):9-13(朱岩,彭振英,范仲学,张斌,王成祥,郑玲,毕玉平,2013,花生蛋白激酶基因Ah STK的克隆与序列分析,山东农业科学,45(1):9-13)