基于转录组金银花WRKY转录因子的挖掘与分析
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摘要
WRKY转录因子是植物抗逆应答过程中最为常见的蛋白家族。为了挖掘金银花抗寒相关的WRKY基因,本研究基于已有的转录组数据,利用植物转录因子数据库和BioEdit对WRKY进行筛选及保守结构域分析,对筛选出的WRKY转录因子利用ExPASy进行理化性质分析,利用PSORT进行亚细胞定位预测,NetPhos进行磷酸化位点分析并且利用MEME数据库对WRKY基因的氨基酸进行序列分析,运用MEGA软件对筛选的金银花WRKY转录因子基因和21个已知抗寒的WRKY基因进行序列同源比对,并构建系统进化树。结果表明:金银花中筛选的30个WRKY基因的蛋白为85～721个氨基酸,理论等电点的范围为4.85到9.76。所有LjWRKY基因编码的蛋白中只有LjWRKY6为稳定蛋白,其余均属于不稳定蛋白,所有蛋白均属于亲水性蛋白。亚细胞定位预测结果,LjWRKY所有蛋白定位于细胞核。除LjWRKY23不具有Try磷酸化位点,其它所有氨基酸均具有Ser、Thr和Try磷酸化位点。LjWRKY蛋白比较保守,WRKYGQK没有发生任何变异,且包含三类WRKY转录因子。系统进化树发现,有12个WRKY基因与已知抗寒基因有同源性,推测其参与了金银花低温逆境下的反应。研究结果可以通过筛选的WRKY基因,进行进一步的转基因验证,进而为植物抗寒分子育种提供分子基础。
WRKY transcrip tion factor is the most common protein family in the process of stress response in plants. In order to explore the WRKY genes of Lonicera japonica related to cold resistance, based on the existing transcriptome data, WRKY was screened using the plant transcription factor database and BioEdit, and the conserved domain analysis was performed. Physicochemical properties of the screened WRKY transcription factors were analyzed using ExPASy. Subcellular localization was predicted by PSORT, NetPhos was used to analyze phosphorylation site analysis and the MEME database was used for sequence analysis of WRKY gene amino acids, the screened Lonicera japonica WRKY transcription factor genes and 21 known cold-resistant WRKY genes were homologous compared by MEGA software, and a phylogenetic tree was constructed. The results showed that the 30 WRKY genes in the Lonicera japonica were 85～721 amino acids and the theoretical isoelectric point ranged from 4.85 to 9.76. Of all the proteins encoded by LJ WRKY gene, only LjWRKY6 was a stable protein, and the rest were all unstable proteins, and all proteins belonged to hydrophilic protein. The subcellular localization predicted that all LjWRKY proteins were located in the nucleus. Except that LjWRKY23 did not have Try Ph acidification sites, all other amino acids had Ser, Thr and Try phosphorylation sites. LjWRKY protein was relatively conserved, WRKYGQK did not have any mutation, and contained three types of WRKY transcription factors. Phylogenetic trees revealed that 12 WRKY genes shared homology with known cold-resistant genes, and it was speculated that they participated in the reaction of Lonicera japonica under low temperature stress. The results of the study could be further verified by screening WRKY genes, which would provide a molecular basis for plant cold-resistant molecular breeding.
WRKY transcrip tion factor is the most common protein family in the process of stress response in plants. In order to explore the WRKY genes of Lonicera japonica related to cold resistance, based on the existing transcriptome data, WRKY was screened using the plant transcription factor database and BioEdit, and the conserved domain analysis was performed. Physicochemical properties of the screened WRKY transcription factors were analyzed using ExPASy. Subcellular localization was predicted by PSORT, NetPhos was used to analyze phosphorylation site analysis and the MEME database was used for sequence analysis of WRKY gene amino acids, the screened Lonicera japonica WRKY transcription factor genes and 21 known cold-resistant WRKY genes were homologous compared by MEGA software, and a phylogenetic tree was constructed. The results showed that the 30 WRKY genes in the Lonicera japonica were 85～721 amino acids and the theoretical isoelectric point ranged from 4.85 to 9.76. Of all the proteins encoded by LJ WRKY gene, only LjWRKY6 was a stable protein, and the rest were all unstable proteins, and all proteins belonged to hydrophilic protein. The subcellular localization predicted that all LjWRKY proteins were located in the nucleus. Except that LjWRKY23 did not have Try Ph acidification sites, all other amino acids had Ser, Thr and Try phosphorylation sites. LjWRKY protein was relatively conserved, WRKYGQK did not have any mutation, and contained three types of WRKY transcription factors. Phylogenetic trees revealed that 12 WRKY genes shared homology with known cold-resistant genes, and it was speculated that they participated in the reaction of Lonicera japonica under low temperature stress. The results of the study could be further verified by screening WRKY genes, which would provide a molecular basis for plant cold-resistant molecular breeding.
引文
Besseau S.,Li J.,and Palva E.T.,2012,WRKY54 and WRKY70co-operate as negative regulators of leaf senescence in Arabidopsis thaliana,J.Exp.Bot.63(7):2667-2679
Cheng Y.,Yao Z.P.,Ruan M.Y.,Li Z.M.,Ye Q.J.,Wang R.Q.,Zhou G.Z.,Yang Y.J.,and Wan H.J.,2016,Function of the WRKY transcription factor in plant responses to biotic/abiotic stresses,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(11):3214-3223(程远,姚祝平,阮美颖,李志邈,叶青静,王荣青,周国治,杨悦俭,万红建,2016,WRKY转录因子在植物逆境应答中的功能,分子植物育种,14(11):3214-3223)
Esser D.,Hoffmann L.,Pham T.K.,Br覿sen C.,Qiu W.,Wright P.C.,Albers S.V.,and Siebers B.,2016,Protein phosphorylation and its role in archaeal signal transduction,Fems Microbiol.Rev.,40(5):625-647
Eulgem T.,Rushton P.J.,Robatzek S.,and Somssich I.E.,2000,The WRKY superfamily of plant transcription factors,Trends Plant Sci.,5(5):199-206
Giacomelli J.I.,Ribichich K.F.,Dezar C.A.,and Chan R.L.,2010,Expression analyses indicate the involvement of sunflower WRKY transcription factors in stress responses,and phylogenetic reconstructions reveal the existence of a novel clade in the Asteraceae,Plant Sci.,178(4):398-410
Jia Q.,Wu M.Y.,Liang K.J.,Sun X.L.,and Lin W.X.,2014,Advances in applications of genomics in stress resistance studies of crops,Zhongguo Sheng tai Nongye Xuebao(Chinese Journal of Eco-Agriculture),22(4):375-385(贾琪,吴名耀,梁康迳,孙新立,林文雄,2014,基因组学在作物抗逆性研究中的新进展,中国生态农业学报,22(4):375-385)
Li J.,Besseau S.,T觟r觟nen P.,Sipari N.,Kollist H.,Holm L.,and Palva E.T.,2013,Defense-related transcription factors WR-KY 70 and WRKY 54 modulate osmotic stress tolerance by regulating stomatal aperture in Arabidopsis,New Phytologist,200(2):457-720
Li L.,Xie B.Y.,Dai X.F.,and Yang Y.H.,2005,WRKY transcription factors and their roles in plant defense responses,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),3(3):401-408(李蕾,谢丙炎,戴小枫,杨宇红,2005,WRKY转录因子及其在植物防御反应中的作用,分子植物育种,3(3):401-408)
Li Y.,2015,Cloning,expression and transcriptional activity analysis of eight wheat WRKY transcription factor,Thesis for M.S.,Huazhong University of Science and Technology,Supervisor:Yang G.X.,pp.38-58(栗颖,2015,8个小麦WRKY转录因子基因的克隆、表达及转录活性分析,硕士学位论文,华中科技大学,导师:杨广笑,pp.38-58)
Liu H.,Li D.J.,and Deng Z.,2014,Advances in research of transcriptional regulatory network in response to cold stress in plants,Zhongguo Nongye Kexue(Chinese Agricultural Sciences),47(18):3523-3533(刘辉,李德军,邓治,2014,植物应答低温胁迫的转录调控网络研究进展,中国农业科学,47(18):3523-3533)
Qiu B.,2013,The cloning and functional study of AaWRKY 3transcription factor in Artemisia annua L.,Thesis for M.S.,Shanghai Jiaotong University,Supervisor:Tang K.X.,pp.48-51(邱波,2013,青蒿AaWRKY 3转录因子的克隆及功能分析,硕士学位论文,上海交通大学,导师:唐克轩,pp.48-51)
Sang X.H.,Wu Z.Y.,Huang C.L.,and Zhang L.S.,2004,Progresses on transcription factors related to plant stress-tolerance,Zhiwu Xuebao(Chinese Bulletin of Botany),21(6):700-708(桑新华,吴忠义,黄丛林,张潞生,2004,植物逆境抗性相关转录因子的研究进展,植物学报,21(6):700-708)
Sun S.H.,and Yu D.Q.,2016,WRKY transcription factors in regulation of stress response in plant,Shengwu Jishu Tongbao(Biotechnology Bulletin),32(10):66-76(孙淑豪,余迪求,2016,WRKY转录因子家族调控植物逆境胁迫响应,生物技术通报,32(10):66-76)
Wang F.,Hou X.,Tang J.,Wang Z.,Wang S.,Jiang F.,and Li Y.,2012,A novel cold-inducible gene from Pak-choi(Brassica campestris ssp chinensis),BcWRKY46,enhances the cold,salt and dehydration stress tolerance in transgenic tobacco,Mol.Biol.Rep.,39(4):4553-4564
Wu L.T.,Du C.F.,Zhang M.Q.,Zhou L.,and Han H.S.,2013,The structure and function of WRKY transcription factors in abiotic and biotic stress,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),11(4):634-638(伍林涛,杜才富,张敏琴,周凌,韩宏仕,2013,WRKY转录因子的结构及其在植物抗逆境胁迫中的功能,分子植物育种,11(4):634-638)
Xie Z.W.,Wang L.J.,Chen M.Y.,Wang J.,Su Y.J.,Yang X.S.,and Cao Q.H.,2016,Studies on WRKY transcription factors and their biological functions in palnts,Zhongguo Nongye Keji Daobao(Chinese Agricultural Science and Technology Review),18(3):46-54(谢政文,王连军,陈锦洋,王娇,苏一钧,杨新笋,曹清河,2016,植物WRKY转录因子及其生物学功能研究进展,中国农业科技导报,18(3):46-54)
Zhai Q.L.,2013,Cloning and functional analysis of HbWRKY7and HbWRKY11 in Hevea brasiliensis,Thesis for M.S.,College of Agriculture,Hainan University,Supervisor:Li W.G.,pp.70-73(翟琪麟,2013,橡胶树HbWRKY7和HbWRKY11的克隆及功能研究,硕士学位论文,海南大学农学院,导师:李维国,pp.70-73)
Zhang F.,Yin J.L.,Guo Y.Q.,and Yue Y.L.,2018,Research advance on WRKY transcription factors,Shengwu Jishu Tongbao(Biotechnology Bulletin),34(1):40-48(张凡,尹俊龙,郭瑛琪,岳艳玲,2018,WRKY转录因子的研究进展,生物技术通报,34(1):40-48)
Zhang Y.,2012,Expression characteristics and functional analysis of Cs WRKY 46 and Cs WRKY 21 response to chilling in cucumber,Dissertation for Ph.D.,Institute of Vegetables and Flowers,Chinese Academy of Agricultural Sciences,Supervisor:Jiang W.J.,pp.65-66(张颖,2012,黄瓜低温胁迫应答转录因子Cs WRKY 46和Cs WRKY 21的表达特征与功能分析,博士学位论文,中国农业科学院蔬菜花卉研究所,导师:蒋卫杰,pp.65-66)
Zhong G.M.,Wu L.T.,Wang J.M.,Yang Y.,and Li X.F.,2012,Subcellular localization and expression analysis of transcription factor At WRKY 28 under biotic stresses,Zhongguo Nongye Keji Daobao(Journal of Agricultural Science and Technology),14(5):57-63(钟贵买,伍林涛,王健美,杨毅,李旭锋,2012,转录因子At WRKY 28亚细胞定位及在非生物胁迫下的表达分析,中国农业科技导报,14(5):57-63)
Zhou Q.Y.,Tian A.G.,Zou H.F.,Xie Z.M.,Lei G.,Huang J.,Wang C.M.,Wang H.W.,Zhang J.S.,and Chen S.Y.,2008,Soybean WRKY-type transcription factor genes,GmWRKY13,GmWRKY21,and GmWRKY54,confer differential tolerance to abiotic stresses in transgenic Arabidopsis,plants,Plant Biotechnol.J.,6(5):486-503
Zhu X.,Liu S.,Chen M.,Qin L.,Kong L.,and Xia G.,2013,WR-KY transcription factors in wheat and their induction by biotic and abiotic stress,Plant Mol.Biol.Rep.,31(5):1053-1067
Zou C.S.,Jiang W.B.,and Yu D.,2010,Male gametophyte-specific WRKY34 transcription factor mediates cold sensitivity of mature pollen in Arabidopsis,J.Exp.Bot.,61(14):3901-3914
Cheng Y.,Yao Z.P.,Ruan M.Y.,Li Z.M.,Ye Q.J.,Wang R.Q.,Zhou G.Z.,Yang Y.J.,and Wan H.J.,2016,Function of the WRKY transcription factor in plant responses to biotic/abiotic stresses,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(11):3214-3223(程远,姚祝平,阮美颖,李志邈,叶青静,王荣青,周国治,杨悦俭,万红建,2016,WRKY转录因子在植物逆境应答中的功能,分子植物育种,14(11):3214-3223)
Esser D.,Hoffmann L.,Pham T.K.,Br覿sen C.,Qiu W.,Wright P.C.,Albers S.V.,and Siebers B.,2016,Protein phosphorylation and its role in archaeal signal transduction,Fems Microbiol.Rev.,40(5):625-647
Eulgem T.,Rushton P.J.,Robatzek S.,and Somssich I.E.,2000,The WRKY superfamily of plant transcription factors,Trends Plant Sci.,5(5):199-206
Giacomelli J.I.,Ribichich K.F.,Dezar C.A.,and Chan R.L.,2010,Expression analyses indicate the involvement of sunflower WRKY transcription factors in stress responses,and phylogenetic reconstructions reveal the existence of a novel clade in the Asteraceae,Plant Sci.,178(4):398-410
Jia Q.,Wu M.Y.,Liang K.J.,Sun X.L.,and Lin W.X.,2014,Advances in applications of genomics in stress resistance studies of crops,Zhongguo Sheng tai Nongye Xuebao(Chinese Journal of Eco-Agriculture),22(4):375-385(贾琪,吴名耀,梁康迳,孙新立,林文雄,2014,基因组学在作物抗逆性研究中的新进展,中国生态农业学报,22(4):375-385)
Li J.,Besseau S.,T觟r觟nen P.,Sipari N.,Kollist H.,Holm L.,and Palva E.T.,2013,Defense-related transcription factors WR-KY 70 and WRKY 54 modulate osmotic stress tolerance by regulating stomatal aperture in Arabidopsis,New Phytologist,200(2):457-720
Li L.,Xie B.Y.,Dai X.F.,and Yang Y.H.,2005,WRKY transcription factors and their roles in plant defense responses,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),3(3):401-408(李蕾,谢丙炎,戴小枫,杨宇红,2005,WRKY转录因子及其在植物防御反应中的作用,分子植物育种,3(3):401-408)
Li Y.,2015,Cloning,expression and transcriptional activity analysis of eight wheat WRKY transcription factor,Thesis for M.S.,Huazhong University of Science and Technology,Supervisor:Yang G.X.,pp.38-58(栗颖,2015,8个小麦WRKY转录因子基因的克隆、表达及转录活性分析,硕士学位论文,华中科技大学,导师:杨广笑,pp.38-58)
Liu H.,Li D.J.,and Deng Z.,2014,Advances in research of transcriptional regulatory network in response to cold stress in plants,Zhongguo Nongye Kexue(Chinese Agricultural Sciences),47(18):3523-3533(刘辉,李德军,邓治,2014,植物应答低温胁迫的转录调控网络研究进展,中国农业科学,47(18):3523-3533)
Qiu B.,2013,The cloning and functional study of AaWRKY 3transcription factor in Artemisia annua L.,Thesis for M.S.,Shanghai Jiaotong University,Supervisor:Tang K.X.,pp.48-51(邱波,2013,青蒿AaWRKY 3转录因子的克隆及功能分析,硕士学位论文,上海交通大学,导师:唐克轩,pp.48-51)
Sang X.H.,Wu Z.Y.,Huang C.L.,and Zhang L.S.,2004,Progresses on transcription factors related to plant stress-tolerance,Zhiwu Xuebao(Chinese Bulletin of Botany),21(6):700-708(桑新华,吴忠义,黄丛林,张潞生,2004,植物逆境抗性相关转录因子的研究进展,植物学报,21(6):700-708)
Sun S.H.,and Yu D.Q.,2016,WRKY transcription factors in regulation of stress response in plant,Shengwu Jishu Tongbao(Biotechnology Bulletin),32(10):66-76(孙淑豪,余迪求,2016,WRKY转录因子家族调控植物逆境胁迫响应,生物技术通报,32(10):66-76)
Wang F.,Hou X.,Tang J.,Wang Z.,Wang S.,Jiang F.,and Li Y.,2012,A novel cold-inducible gene from Pak-choi(Brassica campestris ssp chinensis),BcWRKY46,enhances the cold,salt and dehydration stress tolerance in transgenic tobacco,Mol.Biol.Rep.,39(4):4553-4564
Wu L.T.,Du C.F.,Zhang M.Q.,Zhou L.,and Han H.S.,2013,The structure and function of WRKY transcription factors in abiotic and biotic stress,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),11(4):634-638(伍林涛,杜才富,张敏琴,周凌,韩宏仕,2013,WRKY转录因子的结构及其在植物抗逆境胁迫中的功能,分子植物育种,11(4):634-638)
Xie Z.W.,Wang L.J.,Chen M.Y.,Wang J.,Su Y.J.,Yang X.S.,and Cao Q.H.,2016,Studies on WRKY transcription factors and their biological functions in palnts,Zhongguo Nongye Keji Daobao(Chinese Agricultural Science and Technology Review),18(3):46-54(谢政文,王连军,陈锦洋,王娇,苏一钧,杨新笋,曹清河,2016,植物WRKY转录因子及其生物学功能研究进展,中国农业科技导报,18(3):46-54)
Zhai Q.L.,2013,Cloning and functional analysis of HbWRKY7and HbWRKY11 in Hevea brasiliensis,Thesis for M.S.,College of Agriculture,Hainan University,Supervisor:Li W.G.,pp.70-73(翟琪麟,2013,橡胶树HbWRKY7和HbWRKY11的克隆及功能研究,硕士学位论文,海南大学农学院,导师:李维国,pp.70-73)
Zhang F.,Yin J.L.,Guo Y.Q.,and Yue Y.L.,2018,Research advance on WRKY transcription factors,Shengwu Jishu Tongbao(Biotechnology Bulletin),34(1):40-48(张凡,尹俊龙,郭瑛琪,岳艳玲,2018,WRKY转录因子的研究进展,生物技术通报,34(1):40-48)
Zhang Y.,2012,Expression characteristics and functional analysis of Cs WRKY 46 and Cs WRKY 21 response to chilling in cucumber,Dissertation for Ph.D.,Institute of Vegetables and Flowers,Chinese Academy of Agricultural Sciences,Supervisor:Jiang W.J.,pp.65-66(张颖,2012,黄瓜低温胁迫应答转录因子Cs WRKY 46和Cs WRKY 21的表达特征与功能分析,博士学位论文,中国农业科学院蔬菜花卉研究所,导师:蒋卫杰,pp.65-66)
Zhong G.M.,Wu L.T.,Wang J.M.,Yang Y.,and Li X.F.,2012,Subcellular localization and expression analysis of transcription factor At WRKY 28 under biotic stresses,Zhongguo Nongye Keji Daobao(Journal of Agricultural Science and Technology),14(5):57-63(钟贵买,伍林涛,王健美,杨毅,李旭锋,2012,转录因子At WRKY 28亚细胞定位及在非生物胁迫下的表达分析,中国农业科技导报,14(5):57-63)
Zhou Q.Y.,Tian A.G.,Zou H.F.,Xie Z.M.,Lei G.,Huang J.,Wang C.M.,Wang H.W.,Zhang J.S.,and Chen S.Y.,2008,Soybean WRKY-type transcription factor genes,GmWRKY13,GmWRKY21,and GmWRKY54,confer differential tolerance to abiotic stresses in transgenic Arabidopsis,plants,Plant Biotechnol.J.,6(5):486-503
Zhu X.,Liu S.,Chen M.,Qin L.,Kong L.,and Xia G.,2013,WR-KY transcription factors in wheat and their induction by biotic and abiotic stress,Plant Mol.Biol.Rep.,31(5):1053-1067
Zou C.S.,Jiang W.B.,and Yu D.,2010,Male gametophyte-specific WRKY34 transcription factor mediates cold sensitivity of mature pollen in Arabidopsis,J.Exp.Bot.,61(14):3901-3914
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