辣椒CaWRKY8基因克隆及胁迫下的表达分析
|
摘要
为了揭示辣椒WRKY基因功能,以辣椒PI201234为实验材料,克隆得到WRKY基因全长1 647bp的cDNA序列,命名为CaWRKY8。生物信息学分析表明,该基因含有一个1 647bp完整开放阅读框(ORF),编码548个氨基酸残基。氨基酸序列分析显示,CaWRKY8编码的蛋白含有2个WRKY结构域,属于Group I。氨基酸序列比对结果表明,CaWRKY8与辣椒WRKY25、马铃薯WRKY、番茄基因组中预测的WRKY26、烟草基因组中预测的WRKY33和猕猴桃WRKY的氨基酸序列之间均具有高度的保守性。实时荧光定量分析表明,CaWRKY8受盐、高温、干旱和辣椒疫霉菌诱导表达;其中CaWRKY8的表达量在盐和干旱处理下3h达到峰值,分别是对照的2.38倍和121.10倍,在高温和疫霉菌处理下12h达到峰值,分别是对照的6.12和6.81倍。以上研究结果表明,CaWRKY8基因在辣椒响应胁迫进程中发挥着重要作用。
In order to reveal the function of WRKYgene in pepper,we cloned the full-length 1 647 bp cDNA of WRKY gene from pepper PI201234 and named as CaWRKY8.Bioinformatics analysis indicated that the gene contains a 1 647 bp complete open reading frame(ORF)box encoding 548 amino acid residues.Amino acid sequence analysis revealed that the CaWRKY8 encoded protein contains two WRKY domains belonging to Group I.The amino acid sequence alignment showed that there was a high degree of conservation between CaWRKY8 and the amino acid sequences of pepper WRKY25,potato WRKY,tomato predicted WRKY26,tobacco predicted WRKY33 and kiwi WRKY.Real-time fluorescence quantitative analysis showed that CaWRKY8 was induced by salt,high temperature,drought and Phytophthora capsici,and CaWRKY8 peaked at 3 hunder salt and drought treatments by 2.38 and 121.10 times of control,respectively.Under high temperature and P.capsici treatment,the peak reached at 12 h,which was 6.12 and6.81 times of the control,respectively.The above results indicate that CaWRKY8 played an important role in the response of pepper to stress.
In order to reveal the function of WRKYgene in pepper,we cloned the full-length 1 647 bp cDNA of WRKY gene from pepper PI201234 and named as CaWRKY8.Bioinformatics analysis indicated that the gene contains a 1 647 bp complete open reading frame(ORF)box encoding 548 amino acid residues.Amino acid sequence analysis revealed that the CaWRKY8 encoded protein contains two WRKY domains belonging to Group I.The amino acid sequence alignment showed that there was a high degree of conservation between CaWRKY8 and the amino acid sequences of pepper WRKY25,potato WRKY,tomato predicted WRKY26,tobacco predicted WRKY33 and kiwi WRKY.Real-time fluorescence quantitative analysis showed that CaWRKY8 was induced by salt,high temperature,drought and Phytophthora capsici,and CaWRKY8 peaked at 3 hunder salt and drought treatments by 2.38 and 121.10 times of control,respectively.Under high temperature and P.capsici treatment,the peak reached at 12 h,which was 6.12 and6.81 times of the control,respectively.The above results indicate that CaWRKY8 played an important role in the response of pepper to stress.
引文
[1]KUNKEL B N,BROOKS D M.Cross talk between signaling pathways in pathogen defense[J].Current Opinion in Plant Biology,2002,5(4):325-331.
[2]SINGH K,FOLEY R C,OATE-SNCHEZ L.Transcription factors in plant defense and stress responses[J].Current Opinion in Plant Biology,2002,5(5):430-436.
[3]MAHALINGAM R,GOMEZ-BUITRAGO A,ECKARDTN,et al.Characterizing the stress/defense transcriptome of Arabidopsis[J].Genome Biology,2003,4(3):R20.
[4]肖翔.辣椒一个新的WRKY转录因子的克隆及其功能初步分析[D].福州:福建农林大学,2011.
[5]EULGEM T,RUSHTON P J,ROBATZEK S,et al.The WRKY superfamily of plant transcription factors[J].Trends in Plant Science,2000,5(5):199-206.
[6]ISHIGURO S,NAKAMURA K.Characterization of a cDNAencoding a novel DNA-binding protein,SPF1,that recognizes SP8sequences in the 5′upstream regions of genes coding for sporamin andβ-amylase from sweet potato[J].Molecular&General Genetics,1994,244(6):563-571.
[7]江腾,林勇祥,刘雪,等.苜蓿全基因组WRKY转录因子基因的分析[J].草业学报,2011,20(3):211-218.JIANG T,LIN Y X,LIU X,et al.Genome-wide analysis of the WRKY transcription factor family in Medicago truncatula[J].Acta Prataculturae Sinica,2011,20(3):211-218.
[8]LING J,JIANG W J,ZHANG Y,et al.Genome-wide analysis of WRKY gene family in Cucumis sativus[J].BMC Genomics,2011,12:471.
[9]HUANG S X,GAO Y F,LIU J K,et al.Genome-wide analysis of WRKY transcription factors in Solanum lycopersicum[J].Molecular Genetics and Genomics,2012,287(6):495-513.
[10]许瑞瑞,张世忠,曹慧,等.苹果WRKY转录因子家族基因生物信息学分析[J].园艺学报,2012,39(10):2 049-2 060.XU R R,ZHANG S Z,CAO H,et al.Bioinformatics analysis of WRKY transcription factor genes family in apple[J].Acta Horticulturae Sinica,2012,39(10):2 049-2 060.
[11]邹智.蓖麻WRKY转录因子的全基因组鉴定及其进化分析[J].中国油料作物学报,2013,35(1):36-42.ZOU Z.Genome-wide identification and phylogenetic analysis of WRKY transcription factor family in castor bean[J].Chinese Journal of Oil Crop Sciences,2013,35(1):36-42.
[12]JIANG Y Z,DUAN Y J,YIN J,et al.Genome-wide identification and characterization of the Populus WRKY transcription factor family and analysis of their expression in response to biotic and abiotic stresses[J].Journal of Experimental Botany,2014,65(22):6 629-6 644.
[13]DING M Q,CHEN J D,JIANG Y R,et al.Genome-wide investigation and transcriptome analysis of the WRKY gene family in Gossypium[J].Molecular Genetics and Genomics Mgg,2015,290(1):151-171.
[14]MA J C,LU J,XU J M,et al.Genome-wide identification of WRKY genes in the desert poplar Populus euphratica and adaptive evolution of the genes in response to salt stress[J].Evolutionary Bioinformatics Online,2015,11(Suppl 1):47-55.
[15]BANERJEE A,ROYCHOUDHURY A.WRKY proteins:signaling and regulation of expression during abiotic stress responses[J].The Scientific World Journal,2015,DOI:10.1155120151807560.
[16]DANG F F,WANG Y N,YU L,et al.CaWRKY40,a WRKY protein of pepper,plays an important role in the regulation of tolerance to heat stress and resistance to Ralstonia solanacearum infection[J].Plant Cell&Environment,2013,36(4):757-774.
[17]WANG Y N,DANG F F,LIU Z Q,et al.CaWRKY58,encoding agroup I WRKY transcription factor of Capsicum annuum,negatively regulates resistance to Ralstonia solanacearuminfection[J].Molecular Plant Pathology,2013,14(2):131-144.
[18]DANG F F,WANG Y N,SHE J J,et al.Overexpression of CaWRKY27,a subgroup IIe WRKY transcription factor of Capsicum annuum,positively regulates tobacco resistance to Ralstonia solanacearum,infection[J].Physiologia Plantarum,2014,150(3):397-411.
[19]PARK C J,SHIN Y C,LEE B J,et al.A hot pepper gene encoding WRKY transcription factor is induced during hypersensitive response to tobacco mosaic virus,and Xanthomonas campestris[J].Planta,2006,223(2):168-179.
[20]李淑敏,茆振川,李蕾,等.辣椒抗根结线虫相关WRKY基因的分离[J].园艺学报,2008,35(10):1 467-1 472.LI S M,MAO Z C,LI L,et al.Isolation of WRKY genes in the incompatible interaction between Meloidogyne incognita and Capsicum annuum[J].Acta Horticulturae Sinica,2008,35(10):1 467-1 472.
[21]王育娜,贺俐,曹蕾,等.辣椒推定WRKY基因cDNA的生物信息学分析[J].中国农学通报,2008,24(11):93-96.WANG Y N,HE L,CAO L,et al.Bioinformatic analysis of apepper putative WRKY transcriptional factor[J].Chinese Agricultural Science Bulletin,2008,24(11):93-96.
[22]张春秋.辣椒Me3基因介导抗根结线虫WRKY基因CaRKNIF1的分离及其功能分析[D].北京:中国农业科学院,2010.
[23]刁卫平,王述彬,刘金兵,等.辣椒WRKY转录因子的鉴别及进化分析[J].华北农学报,2014,29(6):45-51.DIAO W P,WANG S B,LIU J B,et al.Identification and phylogenetics analysis of WRKY transcription factors family in pepper[J].Acta Agriculturae Boreali-Sinica,2014,29(6):45-51.
[24]WANG S B,LIU K W,DIAO W P,et al.Evaluation of appropriate reference genes for gene expression studies in pepper by quantitative real-time PCR[J].Molecular Breeding,2012,30(3):1 393-1 400.
[25]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[J].Plant Biotechnology Journal,2008,6(5):486-503.
[26]EULGEM T,SOMSSICH I E.Networks of WRKY transcription factors in defense signaling[J].Current Opinion in Plant Biology,2007,10(4):366-371.
[27]SANCHEZ-BALLESTA M T,LLUCH Y,GOSALBES MJ,et al.A survey of genes differentially expressed during long-term heat-induced chilling tolerance in citrus fruit[J].Planta,2003,218(1):65-70.
[28]GUO Z,KAN Y,CHEN X,et al.Characterization of a rice WRKY gene whose expression is induced upon pathogen attack and mechanical wounding[J].Acta Botanica Sinica,2004,46(8):955-964.
[29]LIU X Q,BAI X Q,WANG X J,et al.OsWRKY71,a rice transcription factor,is involved in rice defense response[J].Journal of Plant Physiology,2007,164(8):969-979.
[30]王育娜.辣椒WRKY转录因子cDNA的分离与功能鉴定[D].福州:福建农林大学,2008.
[2]SINGH K,FOLEY R C,OATE-SNCHEZ L.Transcription factors in plant defense and stress responses[J].Current Opinion in Plant Biology,2002,5(5):430-436.
[3]MAHALINGAM R,GOMEZ-BUITRAGO A,ECKARDTN,et al.Characterizing the stress/defense transcriptome of Arabidopsis[J].Genome Biology,2003,4(3):R20.
[4]肖翔.辣椒一个新的WRKY转录因子的克隆及其功能初步分析[D].福州:福建农林大学,2011.
[5]EULGEM T,RUSHTON P J,ROBATZEK S,et al.The WRKY superfamily of plant transcription factors[J].Trends in Plant Science,2000,5(5):199-206.
[6]ISHIGURO S,NAKAMURA K.Characterization of a cDNAencoding a novel DNA-binding protein,SPF1,that recognizes SP8sequences in the 5′upstream regions of genes coding for sporamin andβ-amylase from sweet potato[J].Molecular&General Genetics,1994,244(6):563-571.
[7]江腾,林勇祥,刘雪,等.苜蓿全基因组WRKY转录因子基因的分析[J].草业学报,2011,20(3):211-218.JIANG T,LIN Y X,LIU X,et al.Genome-wide analysis of the WRKY transcription factor family in Medicago truncatula[J].Acta Prataculturae Sinica,2011,20(3):211-218.
[8]LING J,JIANG W J,ZHANG Y,et al.Genome-wide analysis of WRKY gene family in Cucumis sativus[J].BMC Genomics,2011,12:471.
[9]HUANG S X,GAO Y F,LIU J K,et al.Genome-wide analysis of WRKY transcription factors in Solanum lycopersicum[J].Molecular Genetics and Genomics,2012,287(6):495-513.
[10]许瑞瑞,张世忠,曹慧,等.苹果WRKY转录因子家族基因生物信息学分析[J].园艺学报,2012,39(10):2 049-2 060.XU R R,ZHANG S Z,CAO H,et al.Bioinformatics analysis of WRKY transcription factor genes family in apple[J].Acta Horticulturae Sinica,2012,39(10):2 049-2 060.
[11]邹智.蓖麻WRKY转录因子的全基因组鉴定及其进化分析[J].中国油料作物学报,2013,35(1):36-42.ZOU Z.Genome-wide identification and phylogenetic analysis of WRKY transcription factor family in castor bean[J].Chinese Journal of Oil Crop Sciences,2013,35(1):36-42.
[12]JIANG Y Z,DUAN Y J,YIN J,et al.Genome-wide identification and characterization of the Populus WRKY transcription factor family and analysis of their expression in response to biotic and abiotic stresses[J].Journal of Experimental Botany,2014,65(22):6 629-6 644.
[13]DING M Q,CHEN J D,JIANG Y R,et al.Genome-wide investigation and transcriptome analysis of the WRKY gene family in Gossypium[J].Molecular Genetics and Genomics Mgg,2015,290(1):151-171.
[14]MA J C,LU J,XU J M,et al.Genome-wide identification of WRKY genes in the desert poplar Populus euphratica and adaptive evolution of the genes in response to salt stress[J].Evolutionary Bioinformatics Online,2015,11(Suppl 1):47-55.
[15]BANERJEE A,ROYCHOUDHURY A.WRKY proteins:signaling and regulation of expression during abiotic stress responses[J].The Scientific World Journal,2015,DOI:10.1155120151807560.
[16]DANG F F,WANG Y N,YU L,et al.CaWRKY40,a WRKY protein of pepper,plays an important role in the regulation of tolerance to heat stress and resistance to Ralstonia solanacearum infection[J].Plant Cell&Environment,2013,36(4):757-774.
[17]WANG Y N,DANG F F,LIU Z Q,et al.CaWRKY58,encoding agroup I WRKY transcription factor of Capsicum annuum,negatively regulates resistance to Ralstonia solanacearuminfection[J].Molecular Plant Pathology,2013,14(2):131-144.
[18]DANG F F,WANG Y N,SHE J J,et al.Overexpression of CaWRKY27,a subgroup IIe WRKY transcription factor of Capsicum annuum,positively regulates tobacco resistance to Ralstonia solanacearum,infection[J].Physiologia Plantarum,2014,150(3):397-411.
[19]PARK C J,SHIN Y C,LEE B J,et al.A hot pepper gene encoding WRKY transcription factor is induced during hypersensitive response to tobacco mosaic virus,and Xanthomonas campestris[J].Planta,2006,223(2):168-179.
[20]李淑敏,茆振川,李蕾,等.辣椒抗根结线虫相关WRKY基因的分离[J].园艺学报,2008,35(10):1 467-1 472.LI S M,MAO Z C,LI L,et al.Isolation of WRKY genes in the incompatible interaction between Meloidogyne incognita and Capsicum annuum[J].Acta Horticulturae Sinica,2008,35(10):1 467-1 472.
[21]王育娜,贺俐,曹蕾,等.辣椒推定WRKY基因cDNA的生物信息学分析[J].中国农学通报,2008,24(11):93-96.WANG Y N,HE L,CAO L,et al.Bioinformatic analysis of apepper putative WRKY transcriptional factor[J].Chinese Agricultural Science Bulletin,2008,24(11):93-96.
[22]张春秋.辣椒Me3基因介导抗根结线虫WRKY基因CaRKNIF1的分离及其功能分析[D].北京:中国农业科学院,2010.
[23]刁卫平,王述彬,刘金兵,等.辣椒WRKY转录因子的鉴别及进化分析[J].华北农学报,2014,29(6):45-51.DIAO W P,WANG S B,LIU J B,et al.Identification and phylogenetics analysis of WRKY transcription factors family in pepper[J].Acta Agriculturae Boreali-Sinica,2014,29(6):45-51.
[24]WANG S B,LIU K W,DIAO W P,et al.Evaluation of appropriate reference genes for gene expression studies in pepper by quantitative real-time PCR[J].Molecular Breeding,2012,30(3):1 393-1 400.
[25]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[J].Plant Biotechnology Journal,2008,6(5):486-503.
[26]EULGEM T,SOMSSICH I E.Networks of WRKY transcription factors in defense signaling[J].Current Opinion in Plant Biology,2007,10(4):366-371.
[27]SANCHEZ-BALLESTA M T,LLUCH Y,GOSALBES MJ,et al.A survey of genes differentially expressed during long-term heat-induced chilling tolerance in citrus fruit[J].Planta,2003,218(1):65-70.
[28]GUO Z,KAN Y,CHEN X,et al.Characterization of a rice WRKY gene whose expression is induced upon pathogen attack and mechanical wounding[J].Acta Botanica Sinica,2004,46(8):955-964.
[29]LIU X Q,BAI X Q,WANG X J,et al.OsWRKY71,a rice transcription factor,is involved in rice defense response[J].Journal of Plant Physiology,2007,164(8):969-979.
[30]王育娜.辣椒WRKY转录因子cDNA的分离与功能鉴定[D].福州:福建农林大学,2008.