8个茶树WRKY转录因子基因的克隆与表达分析
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摘要
目的克隆茶树WRKY转录因子(Cs WRKYs)家族中的8个成员,并对其进行生物信息学和非生物胁迫下的表达分析。方法采用实时荧光定量PCR(qRT-PCR)技术从"铁观音"茶树品种中克隆8个WRKY基因,运用生物信息学的方法对8个WRKY基因编码的蛋白进行理化性质分析,同时通过与拟南芥同源基因的比较进行进化树的构建、多序列比对及保守基序分析。采用qRT-PCR方法检测8个WRKY基因在低温、干旱和脱落酸(abscisicacid,ABA)胁迫处理下的表达量。结果 8个WRKY转录因子基因开放阅读框(ORF)长度分别为1 407、2 208、1 302、849、978、879、1 443和810 bp,分别编码468、735、433、282、325、292、480和269个氨基酸,GenBank登录号分别为MG298951、MG298952、MG298955、MG298956、MG298957、MG298959、MG298960和MG298963。系统进化树及序列比对分析显示,8个CsWRKYs可以分成了2个大组;除CsWRKY39缺少锌指结构外,其他CsWRKYs均含有WRKYGQK保守七肽结构域及锌指结构组成的WRKY结构域。非生物胁迫下的表达模式显示,CsWRKYs基因在低温、干旱和ABA胁迫处理下均有表达且表达受到不同程度的诱导;CsWRKY2、CsWRKY21、CsWRKY23、CsWRKY44和CsWRKY65基因在低温处理后表达量上调超过2,显著响应低温胁迫;CsWRKY21、CsWRKY23、CsWRKY39和CsWRKY65在干旱胁迫处理12 h及ABA处理6 h时均上调表达。结论克隆获得来自不同组的8个茶树WRKY基因,推测与茶树抗逆密切相关。
Objective To clone eight members of WRKY of transcription factor family in Camellia sinensis, and analyze their bioinformatics and expression under abiotic stress. Methods Eight WRKY transcription factor genes were cloned from Tieguanyin cultivar by RT-PCR, and the physicochemical properties of the eight WRKY protein were analyzed by bioinformatics methods. At the same time, the establishment of phylogenetic tree, comparison of multiple sequences, and analysis of conserved motifs were carried out by comparing WRKY of C. sinensis with homologous genes of Arabidopsis thaliana. Quantitative real-time PCR(qRT-PCR) was used to detect the expression of eight WRKY genes under low temperature, drought, and ABA stress treatment. Results The ORF lengths of eight WRKY genes were 1 407, 2 208, 1 302, 849, 978, 879, 1 443, and 810 bp, encoding 468, 735, 433, 282, 325, 292, 480, and 269 amino acids, respectively. GenBank accession numbers were MG298951, MG298952, MG298955, MG298956, MG298957, MG298959, MG298960, and MG298963, respectively. Phylogenetic tree and sequence alignment analysis showed that eight CsWRKYs could be divided into two groups and contained WRKYGQK conserved domain and zinc finger structures, except that CsWRKY39 lacked zinc finger structure. The expression pattern of CsWRKYs was induced under the condition of low temperature, drought, and ABA stress. The expression of CsWRKY2, Cs WRKY21, CsWRKY23, CsWRKY44 and CsWRKY65 increased to more than 2 after low temperature treatment with significant response to low temperature stress. The expression of CsWRKY21, CsWRKY23, CsWRKY3,9 and CsWRKY65 was up-regulated under 12 h of drought stress and 6 h of ABA treatment. This result indicated that CsWRKYs might be closely related to stress response in C. sinensis. Conclusion Eight CsWRKY genes from different groups were cloned, and this result indicated that CsWRKYs might be closely related to stress response in C. sinensis.
Objective To clone eight members of WRKY of transcription factor family in Camellia sinensis, and analyze their bioinformatics and expression under abiotic stress. Methods Eight WRKY transcription factor genes were cloned from Tieguanyin cultivar by RT-PCR, and the physicochemical properties of the eight WRKY protein were analyzed by bioinformatics methods. At the same time, the establishment of phylogenetic tree, comparison of multiple sequences, and analysis of conserved motifs were carried out by comparing WRKY of C. sinensis with homologous genes of Arabidopsis thaliana. Quantitative real-time PCR(qRT-PCR) was used to detect the expression of eight WRKY genes under low temperature, drought, and ABA stress treatment. Results The ORF lengths of eight WRKY genes were 1 407, 2 208, 1 302, 849, 978, 879, 1 443, and 810 bp, encoding 468, 735, 433, 282, 325, 292, 480, and 269 amino acids, respectively. GenBank accession numbers were MG298951, MG298952, MG298955, MG298956, MG298957, MG298959, MG298960, and MG298963, respectively. Phylogenetic tree and sequence alignment analysis showed that eight CsWRKYs could be divided into two groups and contained WRKYGQK conserved domain and zinc finger structures, except that CsWRKY39 lacked zinc finger structure. The expression pattern of CsWRKYs was induced under the condition of low temperature, drought, and ABA stress. The expression of CsWRKY2, Cs WRKY21, CsWRKY23, CsWRKY44 and CsWRKY65 increased to more than 2 after low temperature treatment with significant response to low temperature stress. The expression of CsWRKY21, CsWRKY23, CsWRKY3,9 and CsWRKY65 was up-regulated under 12 h of drought stress and 6 h of ABA treatment. This result indicated that CsWRKYs might be closely related to stress response in C. sinensis. Conclusion Eight CsWRKY genes from different groups were cloned, and this result indicated that CsWRKYs might be closely related to stress response in C. sinensis.
引文
[1]李书粉,孙富丛,肖理慧,等.植物对非生物胁迫应答的转录因子及调控机制[J].西北植物学报,2006,26(6):1295-1300.
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[3]Eulgem T,Rushton P J,Robatzek S,et al.The WRKYsuperfamily of plant transcription factors[J].Trends Plant Sci,2000,5(5):199-206.
[4]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[J].Plant Mol Biol,2008,68(1/2):81-92.
[5]Wu K L,Guo Z J,Wang H H,et al.The WRKY family of transcription factors in rice and Arabidopsis and their origins[J].Dna Res An Inter J Rapid Publ Reports Genes Genomes,2005,12(1):9-26.
[6]Berri S,Abbruscato P,Faivre-Rampant O,et al.Characterization of WRKY co-regulatory networks in rice and Arabidopsis[J].Bmc Plant Biol,2009,9(1):120.
[7]Guo C,Guo R,Xu X,et al.Evolution and expression analysis of the grape(Vitis vinifera L.)WRKY gene family[J].J Exper Bot,2014,65(6):1513-1528.
[8]Wang H,Wang H,Shao H,et al.Recent advances in utilizing transcription factors to improve plant abiotic stress tolerance by transgenic technology[J].Frontiers Plant Sci,2016,7(248):67.
[9]Ramamoorthy R,Jiang S Y,Kumar N,et al.Acomprehensive transcriptional profiling of the WRKYgene family in rice under various abiotic and phytohormone treatments[J].Plant Cell Physiol,2008,49(6):865-879.
[10]Kayum M A,Jung H J,Park J I,et al.Identification and expression analysis of WRKY family genes under biotic and abiotic stresses in Brassica rapa[J].Mol Genet Genomics,2015,290(1):79-95.
[11]Wang L,Zhu W,Fang L,et al.Genome-wide identification of WRKY family genes and their response to cold stress in Vitis vinifera[J].BMC Plant Biol,2014,14(1):103.
[12]Song Y,Jing S J,Yu D Q,et al.Overexpression of the stress-induced Os WRKY08 improves osmotic stress tolerance in Arabidopsis[J].Chin Sci Bull,2009,54(24):4671-4678.
[13]Zhou Q Y,Tian A G,Zou H F,et al.Soybean WRKY-type transcription factor genes,Gm WRKY13,Gm WRKY21,and Gm WRKY54,confer differential tolerance to abiotic stresses in transgenic Arabidopsis plants[J].Plant Biotechnol J,2008,6(5):486-503.
[14]Ren X,Chen Z,Liu Y,et al.ABO3,a WRKYtranscription factor,mediates plant responses to abscisic acid and drought tolerance in Arabidopsis[J].Plant JCell Mol Biol,2010,63(3):417-429.
[15]王蔚,郭雅玲.茶功能性成分对肺癌作用机制的研究进展[J].中草药,2017,48(17):3654-3661.
[16]胡雅琼,罗理勇,曾亮.茶叶提取物对帕金森病的防治作用研究进展[J].中草药,2014,45(9):1342-1348.
[17]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[J].Mol Genet Genomics,2016,291(1):255-269.
[18]Wang Y,Shu Z,Wang W,et al.CsWRKY2,a novel WRKYgene from Camellia sinensis,is involved in cold and drought stress responses[J].Biol Plant,2016,60(3):1-9.
[19]郭俊红,王伟东,谷星,等.茶树WRKY转录因子基因CsWRKY57的克隆及表达分析[J].茶叶科学,2017,37(4):2201-2209.
[20]Sheng J,Kim C,Zhang Y,et al.The tea tree genome provides insights into tea flavor and independent evolution of caffeine biosynthesis[J].Mol Plant,2017,10(6):866-877.
[21]姚雪倩,陈丹,岳川,等.茶树烯醇酶基因Cs ENO的克隆及其在非生物胁迫中的表达分析[J].园艺学报,2017,44(3):537-546.
[22]王鹏杰,陈丹,俞滢,等.茶树单萜合成酶Cs TPS基因的克隆及表达分析[J].西北植物学报,2017,37(8):1465-1473.
[23]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)[J].Sci Reports,2016,6:19748.
[24]Livak,K J,Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the2(-Delta Delta CT)method[J].Method,2001,25(4):402-408.
[25]Zhang Y,Wang L.The WRKY transcription factor superfamily:Its origin in eukaryotes and expansion in plants[J].BMC Evol Biol,2005,5(1):1-19.
[26]钟贵买,伍林涛,王健美,等.转录因子At WRKY28亚细胞定位及在非生物胁迫下的表达分析[J].中国农业科技导报,2012,14(5):57-63.
[27]李笑,成玉富,杨旭.茄科植物WRKY转录因子的研究进展[J].园艺学报,2017,44(1):170-178.
[28]Agarwal P,Reddy M P,Chikara J.WRKY:its structure,evolutionary relationship,DNA-binding selectivity,role in stress tolerance and development of plants[J].Mol Biol Rep,2011,38(6):3883-3896.
[29]付乾堂,余迪求.拟南芥At WRKY25、At WRKY26和At WRKY33在非生物胁迫条件下的表达分析[J].遗传,2010,32(8):848-856.
[30]李蕾,谢丙炎,戴小枫,等.WRKY转录因子及其在植物防御反应中的作用[J].分子植物育种,2005,3(3):401-408.
[31]Chen L,Yang Y,Liu C,et al.Characterization of WRKYtranscription factors in Solanum lycopersicum reveals collinearity and their expression patterns under cold treatment[J].Biochem Biophys Res Commun,2015,464(3):962-968.
[32]Rushton D L,Tripathi P,Rabara R C,et al.WRKYtranscription factors:Key components in abscisic acid signalling[J].Plant Biot J,2012,10(1):2-11.
[33]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[J].Plant Cell,2010,22(6):1909-1935.
[34]Ulker B,Somssich I E.WRKY transcription factors:from DNA binding towards biological function[J].Curr Opinion Plant Biol,2004,7(5):491-498.
[2]Leetong I,Young R A.Transcription of eukaryotic protein-coding genes[J].Ann Rev Genet Cs,2000,34(1):77-137.
[3]Eulgem T,Rushton P J,Robatzek S,et al.The WRKYsuperfamily of plant transcription factors[J].Trends Plant Sci,2000,5(5):199-206.
[4]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[J].Plant Mol Biol,2008,68(1/2):81-92.
[5]Wu K L,Guo Z J,Wang H H,et al.The WRKY family of transcription factors in rice and Arabidopsis and their origins[J].Dna Res An Inter J Rapid Publ Reports Genes Genomes,2005,12(1):9-26.
[6]Berri S,Abbruscato P,Faivre-Rampant O,et al.Characterization of WRKY co-regulatory networks in rice and Arabidopsis[J].Bmc Plant Biol,2009,9(1):120.
[7]Guo C,Guo R,Xu X,et al.Evolution and expression analysis of the grape(Vitis vinifera L.)WRKY gene family[J].J Exper Bot,2014,65(6):1513-1528.
[8]Wang H,Wang H,Shao H,et al.Recent advances in utilizing transcription factors to improve plant abiotic stress tolerance by transgenic technology[J].Frontiers Plant Sci,2016,7(248):67.
[9]Ramamoorthy R,Jiang S Y,Kumar N,et al.Acomprehensive transcriptional profiling of the WRKYgene family in rice under various abiotic and phytohormone treatments[J].Plant Cell Physiol,2008,49(6):865-879.
[10]Kayum M A,Jung H J,Park J I,et al.Identification and expression analysis of WRKY family genes under biotic and abiotic stresses in Brassica rapa[J].Mol Genet Genomics,2015,290(1):79-95.
[11]Wang L,Zhu W,Fang L,et al.Genome-wide identification of WRKY family genes and their response to cold stress in Vitis vinifera[J].BMC Plant Biol,2014,14(1):103.
[12]Song Y,Jing S J,Yu D Q,et al.Overexpression of the stress-induced Os WRKY08 improves osmotic stress tolerance in Arabidopsis[J].Chin Sci Bull,2009,54(24):4671-4678.
[13]Zhou Q Y,Tian A G,Zou H F,et al.Soybean WRKY-type transcription factor genes,Gm WRKY13,Gm WRKY21,and Gm WRKY54,confer differential tolerance to abiotic stresses in transgenic Arabidopsis plants[J].Plant Biotechnol J,2008,6(5):486-503.
[14]Ren X,Chen Z,Liu Y,et al.ABO3,a WRKYtranscription factor,mediates plant responses to abscisic acid and drought tolerance in Arabidopsis[J].Plant JCell Mol Biol,2010,63(3):417-429.
[15]王蔚,郭雅玲.茶功能性成分对肺癌作用机制的研究进展[J].中草药,2017,48(17):3654-3661.
[16]胡雅琼,罗理勇,曾亮.茶叶提取物对帕金森病的防治作用研究进展[J].中草药,2014,45(9):1342-1348.
[17]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[J].Mol Genet Genomics,2016,291(1):255-269.
[18]Wang Y,Shu Z,Wang W,et al.CsWRKY2,a novel WRKYgene from Camellia sinensis,is involved in cold and drought stress responses[J].Biol Plant,2016,60(3):1-9.
[19]郭俊红,王伟东,谷星,等.茶树WRKY转录因子基因CsWRKY57的克隆及表达分析[J].茶叶科学,2017,37(4):2201-2209.
[20]Sheng J,Kim C,Zhang Y,et al.The tea tree genome provides insights into tea flavor and independent evolution of caffeine biosynthesis[J].Mol Plant,2017,10(6):866-877.
[21]姚雪倩,陈丹,岳川,等.茶树烯醇酶基因Cs ENO的克隆及其在非生物胁迫中的表达分析[J].园艺学报,2017,44(3):537-546.
[22]王鹏杰,陈丹,俞滢,等.茶树单萜合成酶Cs TPS基因的克隆及表达分析[J].西北植物学报,2017,37(8):1465-1473.
[23]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)[J].Sci Reports,2016,6:19748.
[24]Livak,K J,Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the2(-Delta Delta CT)method[J].Method,2001,25(4):402-408.
[25]Zhang Y,Wang L.The WRKY transcription factor superfamily:Its origin in eukaryotes and expansion in plants[J].BMC Evol Biol,2005,5(1):1-19.
[26]钟贵买,伍林涛,王健美,等.转录因子At WRKY28亚细胞定位及在非生物胁迫下的表达分析[J].中国农业科技导报,2012,14(5):57-63.
[27]李笑,成玉富,杨旭.茄科植物WRKY转录因子的研究进展[J].园艺学报,2017,44(1):170-178.
[28]Agarwal P,Reddy M P,Chikara J.WRKY:its structure,evolutionary relationship,DNA-binding selectivity,role in stress tolerance and development of plants[J].Mol Biol Rep,2011,38(6):3883-3896.
[29]付乾堂,余迪求.拟南芥At WRKY25、At WRKY26和At WRKY33在非生物胁迫条件下的表达分析[J].遗传,2010,32(8):848-856.
[30]李蕾,谢丙炎,戴小枫,等.WRKY转录因子及其在植物防御反应中的作用[J].分子植物育种,2005,3(3):401-408.
[31]Chen L,Yang Y,Liu C,et al.Characterization of WRKYtranscription factors in Solanum lycopersicum reveals collinearity and their expression patterns under cold treatment[J].Biochem Biophys Res Commun,2015,464(3):962-968.
[32]Rushton D L,Tripathi P,Rabara R C,et al.WRKYtranscription factors:Key components in abscisic acid signalling[J].Plant Biot J,2012,10(1):2-11.
[33]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[J].Plant Cell,2010,22(6):1909-1935.
[34]Ulker B,Somssich I E.WRKY transcription factors:from DNA binding towards biological function[J].Curr Opinion Plant Biol,2004,7(5):491-498.