茶树CsWRKY6、CsWRKY31和CsWRKY48基因的分离及表达分析
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摘要
以茶树品种‘铁观音’的芽叶为供试材料,采用逆转录-聚合酶链式反应(reverse transcription-polymerase chain reaction, RT-PCR)技术克隆获得3个WRKY基因CsWRKY6(GenBank登录号:MG298953)、CsWRKY31(MG298958)和CsWRKY48(MG298961)。它们的开放阅读框长度分别为1 734、1 299和960 bp,分别编码577、432和319个氨基酸。系统发育树及蛋白结构域分析表明,3个茶树WRKY基因均属于第Ⅱ类WRKY蛋白,都含有高度保守的DNA结合域(WRKYGQK)和锌指结构组成的WRKY结构域,其中Ⅱb亚类的CsWRKY6和CsWRKY31锌指结构模式为C-X_5-C-X_(23)-H-X-H,Ⅱc亚类的CsWRKY48锌指结构模式为C-X_4-C-X_(23)-H-X-H。3个基因在茶树不同组织中均有表达且具有明显的组织特异性,CsWRKY6在老叶中的表达量显著高于其他组织,CsWRKY31在花中的表达量最高,CsWRKY48在根和茎中的表达量显著高于叶、茶花和茶果。蛋白互作预测表明3个基因可能通过与多个基因互作来响应逆境胁迫,荧光定量表达分析显示低温处理能显著上调茶树叶片中3个基因的表达;在干旱处理下CsWRKY31和CsWRKY48表达均显著上调且在12 h后达到最大,外源脱落酸处理后CsWRKY48表达迅速上调,而CsWRKY6和CsWRKY31表达下调。推测这3个基因与茶树抗逆响应密切相关。
Three WRKY genes were cloned by using reverse transcription-polymerase chain reaction(RT-PCR)technique from the tea plant of‘Tieguanyin'cultivar and named as CsWRKY6, CsWRKY31, CsWRKY48 with the GenBank accession numbers of MG298953, MG298958 and MG298961, respectively. Their open reading frames(ORFs) were 1 734, 1 299 and 960 bp long, encoding 577, 432 and 319 amino acids, respectively. Phylogenetic tree and protein domain analysis indicated that all of them belong to class Ⅱ WRKY protein, and contained highly conserved DNA-binding domain of WRKYGQK and zinc finger structures. CsWRKY6 and CsWRKY31 shared the same zinc finger structure model of C-X_5-C-X_(23)-H-X-H, whereas CsWRKY48 belonged to C-X_4-C-X_(23)-H-X-H. The three genes were expressed in different tissues and had obvious tissue specificity in tea plant. The expression level of CsWRKY6 in old leaves was significantly higher than that in other tissues, while the expression level of CsWRKY31 was the highest in flowers and CsWRKY48 had higher expression level in roots and stems than that in leaves, flowers and fruits. Protein interaction prediction indicated that the three genes were involved in the stress response by interacting with multiple genes. Fluorescence quantitative analysis showed that low temperature treatment could significantly up-regulate their transcript abundance in tea plant leaves. Under drought stress, both CsWRKY31 and CsWRKY48 were induced to the maximum after 12 h. The expression level of CsWRKY48 was dramatically up-regulated after exogenous abscisic acid(ABA) treatment, whereas the expression level of CsWRKY6 and CsWRKY31 was repressed. It is revealed that the three genes are closely related to the resistance in tea plants.
Three WRKY genes were cloned by using reverse transcription-polymerase chain reaction(RT-PCR)technique from the tea plant of‘Tieguanyin'cultivar and named as CsWRKY6, CsWRKY31, CsWRKY48 with the GenBank accession numbers of MG298953, MG298958 and MG298961, respectively. Their open reading frames(ORFs) were 1 734, 1 299 and 960 bp long, encoding 577, 432 and 319 amino acids, respectively. Phylogenetic tree and protein domain analysis indicated that all of them belong to class Ⅱ WRKY protein, and contained highly conserved DNA-binding domain of WRKYGQK and zinc finger structures. CsWRKY6 and CsWRKY31 shared the same zinc finger structure model of C-X_5-C-X_(23)-H-X-H, whereas CsWRKY48 belonged to C-X_4-C-X_(23)-H-X-H. The three genes were expressed in different tissues and had obvious tissue specificity in tea plant. The expression level of CsWRKY6 in old leaves was significantly higher than that in other tissues, while the expression level of CsWRKY31 was the highest in flowers and CsWRKY48 had higher expression level in roots and stems than that in leaves, flowers and fruits. Protein interaction prediction indicated that the three genes were involved in the stress response by interacting with multiple genes. Fluorescence quantitative analysis showed that low temperature treatment could significantly up-regulate their transcript abundance in tea plant leaves. Under drought stress, both CsWRKY31 and CsWRKY48 were induced to the maximum after 12 h. The expression level of CsWRKY48 was dramatically up-regulated after exogenous abscisic acid(ABA) treatment, whereas the expression level of CsWRKY6 and CsWRKY31 was repressed. It is revealed that the three genes are closely related to the resistance in tea plants.
引文
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[14]CHEN L G,SONG Y,LI S J,et al.The role of WRKYtranscription factors in plant abiotic stresses.Biochimica et Biophysica Acta(BBA)-Gene Regulatory Mechanisms,2012,1819(2):120-128.
[15]ZOU X L,SEEMANN J R,NEUMAN D,et al.A WRKYgene from creosote bush encodes an activator of the abscisic acid signaling pathway.Journal of Biological Chemistry,2004,279(53):55770-55779.
[16]蒋明,陈贝贝,管铭,等.青花菜转录因子基因BoWRKY2的克隆与表达分析.浙江大学学报(农业与生命科学版),2015,41(2):153-159.JIANG M,CHEN B B,GUAN M,et al.Cloning and expression analysis of a transcription factor gene BoWRKY2from broccoli.Journal of Zhejiang University(Agriculture and Life Sciences),2015,41(2):153-159.(in Chinese with English abstract)
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[18]WANG F,HOU X L,TANG J,et al.A novel cold-inducible gene from Pak-choi(Brassica campestris ssp.chinensis),BcWRKY46,enhances the cold,salt and dehydration stress tolerance in transgenic tobacco.Molecular Biology Reports,2012,39(4):4553-4564.
[19]MA D M,PU G B,LEI C Y,et al.Isolation and characterization of AaWRKY1,an Artemisia annua transcription factor that regulates the amorpha-4,11-diene synthase gene,a key gene of artemisinin biosynthesis.Plant and Cell Physiology,2009,50(12):2146-2161.
[20]DANG F F,WANG Y N,YU L,et al.CaWRKY40,a WRKYprotein of pepper,plays an important role in the regulation of tolerance to heat stress and resistance to Ralstonia solanacearum infection.Plant Cell and Environment,2013,36(4):757-774.
[21]WU Z J,LI X H,LIU Z W,et al.Transcriptome-wide identification of Camellia sinensis WRKY transcription factors in response to temperature stress.Molecular Genetics and Genomics,2016,291:255-269.
[22]罗勇,陈丝,欧淑琼,等.茶树WRKY转录因子的鉴定与分析.分子植物育种,2017,15(10):3932-3940.LUO Y,CHEN S,OU S Q,et al.Identification and analysis of the WRKY transcription factor in tea plant.Molecular Plant Breeding,2017,15(10):3932-3940.(in Chinese with English abstract)
[23]WANG Y,SHU Z,WANG W,et al.CsWRKY2,a novel WRKYgene from Camellia sinensis,is involved in cold and drought stress responses.Biologia Plantarum,2016,60(3):443-451.
[24]郭俊红,王伟东,谷星,等.茶树WRKY转录因子基因Cs‐WRKY57的克隆及表达分析.茶叶科学,2017,37(4):411-419.GUO J H,WANG W D,GU X,et al.Cloning and expression analysis of WRKY transcription factor gene CsWRKY57 in tea plant(Camellia sinensis).Journal of Tea Science,2017,37(4):411-419.(in Chinese with English abstract)
[25]XIA E H,ZHANG H B,SHENG J,et al.The tea tree genome provides insights into tea flavor and independent evolution of caffeine biosynthesis.Molecular Plant,2017,10(6):866-877.
[26]姚雪倩,陈丹,岳川,等.茶树烯醇酶基因CsENO的克隆及其在非生物胁迫中的表达分析.园艺学报,2017,44(3):537-546.YAO X Q,CHEN D,YUE C,et al.Cloning of enolase gene CsENO and its expression analysis under abiotic stress in tea plant.Acta Horticulturae Sinica,2017,44(3):537-546.(in Chinese with English abstract)
[27]王鹏杰,陈丹,俞滢,等.茶树单萜合成酶CsTPS基因的克隆及表达分析.西北植物学报,2017,37(8):1465-1473.WANG P J,CHEN D,YU Y,et al.Clone and expression of monoterpene synthase gene CsTPS in tea plant(Camellia sinensis).Acta Botanica Boreali-Occidentalia Sinica,2017,37(8):1465-1473.(in Chinese with English abstract)
[28]孙美莲,王云生,杨冬青,等.茶树实时荧光定量PCR分析中内参基因的选择.植物学报,2010,45(5):579-587.SUN M L,WANG Y S,YANG D Q,et al.Reference genes for real-time fluorescence quantitative PCR in Camellia sinensis.Chinese Bulletin of Botany,2010,45(5):579-587.(in Chinese with English abstract)
[29]WU Z J,TIAN C,JIANG Q,et al.Selection of suitable reference genes for qRT-PCR normalization during leaf development and hormonal stimuli in tea plant(Camellia sinensis).Scientific Reports,2016,6:19748.
[30]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.Methods,2001,25(4):402-408.
[31]NORIKO Y,MASAHIRO O,YOSHIHIRO Y.The role of the nuclear transport system in cell differentiation.Seminars in Cell and Developmental Biology,2009,20(5):590-599.
[32]ZHENG J Y,ZOU X X,MAO Z C,et al.A novel pepper(Capsicum annuum L.)WRKY Gene,CaWRKY30,is involved in pathogen stress responses.Journal of Plant Biology,2011,54:329-337.
[33]CHEN L,YANG Y,LIU C,et al.Characterization of WRKYtranscription factors in Solanum lycopersicum reveals collinearity and their expression patterns under cold treatment.Biochemical and Biophysical Research Communications,2015,464(3):962-968.
[34]ZHOU Q Y,TIAN A G,ZOU H F,et al.Soybean WRKY-type transcription factor genes,GmWRKY13,GmWRKY21,and GmWRKY54,confer differential tolerance to abiotic stresses in transgenic Arabidopsis plants.Plant Biotechnology Journal,2008,6(5):486-503.
[35]MOON S J,HAN S Y,KIM D Y,et al.Ectopic expression of CaWRKY1,a pepper transcription factor,enhances drought tolerance in transgenic potato plants.Journal of Plant Biology,2014,57:198-207.
[36]SHANG Y,YAN L,LIU Z Q,et al.The Mg-chelatase Hsubunit of Arabidopsis antagonizes a group of WRKYtranscription repressors to relieve ABA-responsive genes of inhibition.Plant Cell,2010,22:1909-1935.
[2]ULKER B,SOMSSICH I E.WRKY transcription factors:from DNA binding towards biological function.Current Opinion in Plant Biology,2004,7(5):491-498.
[3]CIOLKOWSKI I,WANKE D,BIRKENBIHL R P,et al.Studies on DNA-binding selectivity of WRKY transcription factors lend structural clues into WRKY-domain function.Plant Molecular Biology,2008,68(1/2):81-92.
[4]ISHIGURO S,NAKAMURA K.Characterization of a cDNA encoding a novel DNA-binding protein,SPF1,that recognizes SP8 sequences in the 5′upstream regions of genes coding for sporamin andβ-amylase from sweet potato.Molecular and General Genetics,1994,244(6):563-571.
[5]YODA H,OGAWA M,YAMAGUCHI Y,et al.Identification of early-responsive genes associated with the hypersensitive response to tobacco mosaic virus and characterization of a WRKY-type transcription factor in tobacco plants.Molecular and General Genetics,2002,267(2):154-161.
[6]LEVéE V,MAJOR I,LEVASSEUR C,et al.Expression profiling and functional analysis of Populus WRKY23 reveals a regulatory role in defense.New Phytologist,2009,184:48-70.
[7]CHEN H,LAI Z B,SHI J W,et al.Roles of arabidopsis WRKY18,WRKY40 and WRKY60 transcription factors in plant responses to abscisic acid and abiotic stress.BMCPlant Biology,2010,10:281.
[8]YAO D X,ZHANG X Y,ZHAO X H,et al.Transcriptome analysis reveals salt-stress-regulated biological processes and key pathways in roots of cotton(Gossypium hirsutum L.).Genomics,2011,98:47-55.
[9]EULGEM T,RUSHTON P J,ROBATZEK S,et al.The WRKY superfamily of plant transcription factors.Trends in Plant Science,2000,5(5):199-206.
[10]HUANG S X,GAO Y F,LIU J K,et al.Genome-wide analysis of WRKY transcription factors in Solanum lycopersicum.Molecular Genetics and GeTnomics,2012,287(6):495-513.
[11]HE H S,DONG Q,SHAO Y H,et al.Genome-wide survey and characterization of the WRKY gene family in Populus trichocarpa.Plant Cell Reports,2012,31(7):1199-1217.
[12]谷彦冰,冀志蕊,迟福梅,等.苹果WRKY基因家族生物信息学及表达分析.中国农业科学,2015,48(16):3221-3238.GU Y B,JI Z R,CHI F M,et al.Bioinformatics and expression analysis of the WRKY gene family in apple.Scientia Agricultura Sinica,2015,48(16):3221-3238.(in Chinese with English abstract)
[13]PANDEY S P,SOMSSICH I E.The role of WRKYtranscription factors in plant immunity.Plant Physiology,2009,150(4):1648-1655.
[14]CHEN L G,SONG Y,LI S J,et al.The role of WRKYtranscription factors in plant abiotic stresses.Biochimica et Biophysica Acta(BBA)-Gene Regulatory Mechanisms,2012,1819(2):120-128.
[15]ZOU X L,SEEMANN J R,NEUMAN D,et al.A WRKYgene from creosote bush encodes an activator of the abscisic acid signaling pathway.Journal of Biological Chemistry,2004,279(53):55770-55779.
[16]蒋明,陈贝贝,管铭,等.青花菜转录因子基因BoWRKY2的克隆与表达分析.浙江大学学报(农业与生命科学版),2015,41(2):153-159.JIANG M,CHEN B B,GUAN M,et al.Cloning and expression analysis of a transcription factor gene BoWRKY2from broccoli.Journal of Zhejiang University(Agriculture and Life Sciences),2015,41(2):153-159.(in Chinese with English abstract)
[17]RUSHTON D L,TRIPATHI P,RABARA R C,et al.WRKYtranscription factors:key components in abscisic acid signalling.Plant Biotechnology Journal,2012,10(1):2-11.
[18]WANG F,HOU X L,TANG J,et al.A novel cold-inducible gene from Pak-choi(Brassica campestris ssp.chinensis),BcWRKY46,enhances the cold,salt and dehydration stress tolerance in transgenic tobacco.Molecular Biology Reports,2012,39(4):4553-4564.
[19]MA D M,PU G B,LEI C Y,et al.Isolation and characterization of AaWRKY1,an Artemisia annua transcription factor that regulates the amorpha-4,11-diene synthase gene,a key gene of artemisinin biosynthesis.Plant and Cell Physiology,2009,50(12):2146-2161.
[20]DANG F F,WANG Y N,YU L,et al.CaWRKY40,a WRKYprotein of pepper,plays an important role in the regulation of tolerance to heat stress and resistance to Ralstonia solanacearum infection.Plant Cell and Environment,2013,36(4):757-774.
[21]WU Z J,LI X H,LIU Z W,et al.Transcriptome-wide identification of Camellia sinensis WRKY transcription factors in response to temperature stress.Molecular Genetics and Genomics,2016,291:255-269.
[22]罗勇,陈丝,欧淑琼,等.茶树WRKY转录因子的鉴定与分析.分子植物育种,2017,15(10):3932-3940.LUO Y,CHEN S,OU S Q,et al.Identification and analysis of the WRKY transcription factor in tea plant.Molecular Plant Breeding,2017,15(10):3932-3940.(in Chinese with English abstract)
[23]WANG Y,SHU Z,WANG W,et al.CsWRKY2,a novel WRKYgene from Camellia sinensis,is involved in cold and drought stress responses.Biologia Plantarum,2016,60(3):443-451.
[24]郭俊红,王伟东,谷星,等.茶树WRKY转录因子基因Cs‐WRKY57的克隆及表达分析.茶叶科学,2017,37(4):411-419.GUO J H,WANG W D,GU X,et al.Cloning and expression analysis of WRKY transcription factor gene CsWRKY57 in tea plant(Camellia sinensis).Journal of Tea Science,2017,37(4):411-419.(in Chinese with English abstract)
[25]XIA E H,ZHANG H B,SHENG J,et al.The tea tree genome provides insights into tea flavor and independent evolution of caffeine biosynthesis.Molecular Plant,2017,10(6):866-877.
[26]姚雪倩,陈丹,岳川,等.茶树烯醇酶基因CsENO的克隆及其在非生物胁迫中的表达分析.园艺学报,2017,44(3):537-546.YAO X Q,CHEN D,YUE C,et al.Cloning of enolase gene CsENO and its expression analysis under abiotic stress in tea plant.Acta Horticulturae Sinica,2017,44(3):537-546.(in Chinese with English abstract)
[27]王鹏杰,陈丹,俞滢,等.茶树单萜合成酶CsTPS基因的克隆及表达分析.西北植物学报,2017,37(8):1465-1473.WANG P J,CHEN D,YU Y,et al.Clone and expression of monoterpene synthase gene CsTPS in tea plant(Camellia sinensis).Acta Botanica Boreali-Occidentalia Sinica,2017,37(8):1465-1473.(in Chinese with English abstract)
[28]孙美莲,王云生,杨冬青,等.茶树实时荧光定量PCR分析中内参基因的选择.植物学报,2010,45(5):579-587.SUN M L,WANG Y S,YANG D Q,et al.Reference genes for real-time fluorescence quantitative PCR in Camellia sinensis.Chinese Bulletin of Botany,2010,45(5):579-587.(in Chinese with English abstract)
[29]WU Z J,TIAN C,JIANG Q,et al.Selection of suitable reference genes for qRT-PCR normalization during leaf development and hormonal stimuli in tea plant(Camellia sinensis).Scientific Reports,2016,6:19748.
[30]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.Methods,2001,25(4):402-408.
[31]NORIKO Y,MASAHIRO O,YOSHIHIRO Y.The role of the nuclear transport system in cell differentiation.Seminars in Cell and Developmental Biology,2009,20(5):590-599.
[32]ZHENG J Y,ZOU X X,MAO Z C,et al.A novel pepper(Capsicum annuum L.)WRKY Gene,CaWRKY30,is involved in pathogen stress responses.Journal of Plant Biology,2011,54:329-337.
[33]CHEN L,YANG Y,LIU C,et al.Characterization of WRKYtranscription factors in Solanum lycopersicum reveals collinearity and their expression patterns under cold treatment.Biochemical and Biophysical Research Communications,2015,464(3):962-968.
[34]ZHOU Q Y,TIAN A G,ZOU H F,et al.Soybean WRKY-type transcription factor genes,GmWRKY13,GmWRKY21,and GmWRKY54,confer differential tolerance to abiotic stresses in transgenic Arabidopsis plants.Plant Biotechnology Journal,2008,6(5):486-503.
[35]MOON S J,HAN S Y,KIM D Y,et al.Ectopic expression of CaWRKY1,a pepper transcription factor,enhances drought tolerance in transgenic potato plants.Journal of Plant Biology,2014,57:198-207.
[36]SHANG Y,YAN L,LIU Z Q,et al.The Mg-chelatase Hsubunit of Arabidopsis antagonizes a group of WRKYtranscription repressors to relieve ABA-responsive genes of inhibition.Plant Cell,2010,22:1909-1935.