SlCBL1基因在番茄抗灰霉病中的作用
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摘要
以番茄(Solanum lycopersicum L.)品种‘Micro Tom’为试材,分析番茄叶片和果实的灰霉病发病规律,及番茄类钙调磷酸酶B基因(tomato calcineurin B-like gene,SlCBL1)在叶片和果实中的表达变化;比较转SlCBL1基因番茄与对照的叶片和果实的灰霉病发病过程,分析转基因番茄抗病相关转录因子表达变化。结果表明:(1)非转基因番茄中,不同叶龄的叶片均在接种灰霉病4d开始发病;不同发育阶段的果实接种灰霉病后发病时间也不同,其中绿果(花后16~18d)接种5d还未发病,白果(花后34~36d)接种11d开始发病,红果(花后40~42d)接种5d开始发病;SlCBL1基因表达量在番茄叶片中较低,在绿果期和白果期的果实中表达量最高,红果期果实中表达量最低。(2)转SlCBL1基因后,SlCBL1基因的过量表达能够抑制番茄叶片和果实灰霉病发生;同时番茄叶片和果实中几乎所有的抗病转录因子的表达量都上调,其中WRKY转录因子家族基因SlWRKY33和SlWRKY70受到强烈调控。研究说明,SlCBL1基因过量表达能够提高番茄的抗灰霉能力,其主要机理是通过影响抗病相关转录因子进而调控番茄抗灰霉病的能力。
With tomato(Solanum lycopersicum L.)variety‘Micro Tom'as test materials,we analyzed the pathogenesis regularity and SlCBL1 gene expression in tomato leaf and fruit.After inoculation with Botrytis cinerea,we observed the incidence of SlCBL1 gene in tomato leaves and fruits in transgenic tomato and control.We analyzed the expression change of transcription factor related to disease resistance in transgenic tomato and control.The results show that:(1)for Non-gmo tomatoes,the leaves began to attack after inoculation B.cinerea4 days.Green fruit inoculation B.cinerea15 days still no disease.White fruit began to attack after inoculation B.cinerea11 days.Red fruit began to attack after inoculation B.cinerea5 days.SlCBL1gene expression in tomato leaves was lower than in fruit,green fruit and white fruit stage is the highest,the red fruit is the lowest.(2)For genetically modified tomatoes,overexpression of SlCBL1 gene can inhibit the occurrence of B.cinereain tomato leaves and fruits.Almost all the transcrip-tion factor expression was up-regulated in leaves and fruits.WRKY transcription factor family gene SlWRKY33 and SlWRKY70were strongly up-regulated.The results showed that the over expression of SlCBL1 gene could improve the ability of the tomato to resist the B.cinerea.And by influencing the disease resistance related transcription factors to regulation tomato resistance to B.cinerea.
With tomato(Solanum lycopersicum L.)variety‘Micro Tom'as test materials,we analyzed the pathogenesis regularity and SlCBL1 gene expression in tomato leaf and fruit.After inoculation with Botrytis cinerea,we observed the incidence of SlCBL1 gene in tomato leaves and fruits in transgenic tomato and control.We analyzed the expression change of transcription factor related to disease resistance in transgenic tomato and control.The results show that:(1)for Non-gmo tomatoes,the leaves began to attack after inoculation B.cinerea4 days.Green fruit inoculation B.cinerea15 days still no disease.White fruit began to attack after inoculation B.cinerea11 days.Red fruit began to attack after inoculation B.cinerea5 days.SlCBL1gene expression in tomato leaves was lower than in fruit,green fruit and white fruit stage is the highest,the red fruit is the lowest.(2)For genetically modified tomatoes,overexpression of SlCBL1 gene can inhibit the occurrence of B.cinereain tomato leaves and fruits.Almost all the transcrip-tion factor expression was up-regulated in leaves and fruits.WRKY transcription factor family gene SlWRKY33 and SlWRKY70were strongly up-regulated.The results showed that the over expression of SlCBL1 gene could improve the ability of the tomato to resist the B.cinerea.And by influencing the disease resistance related transcription factors to regulation tomato resistance to B.cinerea.
引文
[1]ZHU J K,LIU J,XIONG L.Genetic analysis of salt tolerance in Arabidopsis[J].The Plant Cell,1998,10(7):1 181-1 191.
[2]MARTINEZ-ATIENZA J,JIANG X,GARCIADEBLAS B,et al.Conservation of the salt overly sensitive pathway in rice[J].Plant Physiology,2007,143(2):1 001-1 012.
[3]WANG M Y,GU D,LIU T S,et al.Overexpression of a putative maize calcineurin B-like protein in Arabidopsis confers salt tolerance[J].Plant Molecular Biology,2007,65(6):733-746.
[4]SHI S S,CHEN W,SUNW N.Comparative proteomic analysis of the Arabidopsis cbl1 mutant in response to salt stress[J].Proteomics,2011,11(24):4 712-4 725.
[5]ALBRECHT V,WEINL S,BLAZEVIC D,et al.The calcium sensor CBL1intergrates plant responses to abiotic stresses[J].The Plant Journal,2003,36(4):457-470.
[6]CHEONG Y H,KIM K N,et al.CBL1,a calcium sensor that differentially regulates salt,drought,and cold responses in Arabidopsis[J].The Plant Cell,2003,15(8):1 833-1 845.
[7]马齐军,胡大刚,由春香,等.苹果MdCBL家族基因表达分析及MdCBL1的功能鉴定[J].园艺学报2014,41(6):1 053-1 062.MA Q J,HU D G,YOU C X,et al.Classification and expression analysis of apple MdCBLfamily genes with a functional characterization of MdCBL1[J].Acta Horticulturae Sinica,2014,41(6):1 053-1 062.
[8]韩金龙,李慧,丛郁,等.杜梨CBL1和CBL7基因对非生物逆境的响应[J].果树学报,2014,31(4):529-535.HAN J L,LI H,CONG Y,et al.Comparison of two CBL genes on stress tolerance functions from Pyrus betulaefolia[J].Journal of Fruit Science,2014,31(4):529-535.
[9]许园园,蔺经,李晓刚,等.梨CBL基因家族全基因组序列的鉴定及非生物胁迫下的表达分析[J].中国农业科学,2015,48(4):735-747.XU Y Y,LIN J,LI X G,et al.Identification and expression analysis under abiotic stresses of the CBL gene family in pear[J].Scientia Agricultura Sinica,2015,48(4):735-747.
[10]GAO P,ZHAO P M,WANG J,et al.Co-expression and preferential interaction between two calcineurin B-like proteins and a CBL-interacting protein kinase from cotton[J].Plant Physiology and Biochemistry,2008,46(10):935-940.
[11]赵晋锋,余爱丽,田岗,等.谷子CBL基因鉴定及其在干旱、高盐胁迫下的表达分析[J].作物学报,2013,39(2):360-367.ZHAO J F,YU A L,TIAN G,et al.Identification of CBL genes from foxtail millet(Setariaitalic[L.]Beauv.)and its expression under drought and salt stresses[J].Acta Agronomica Sinica,2013,39(2):360-367.
[12]ZHAO J F,SUN Z F,ZHENG J,et al.Cloning and characterization of a novel CBL-interacting protein kinase from maize[J].Plant Molecular Biology,2009,69(6):661-674.
[13]TOKAS I,PANDEY A,et al.Role of calcium-mediated CBLCIPK network in plant mineral nutrition and abiotic stress[M].Molecular Stress Physiology of Plants,2013:241-261.
[14]DRERUP M M,SCHLUCKING K,HASHIMOTO K,et al.The calcineurin B-like calcium sensors CBL1and CBL9together with their interacting protein kinase CIPK26regulate the Arabidopsis NADPH oxidase RBOHF[J].Molecular Plant,2013,6(2):559-569.
[15]MAHS A,STEINHORST L,HAN J P,et al.The calcineurin B-like Ca2+sensors CBL1and CBL9function in pollen germination and pollen tube growth in Arabidopsis[J].Molecular Plant,2013,6(4):1 149-1 162.
[16]赵统敏,余文贵,赵丽萍,等.番茄抗灰霉病育种研究进展[J].江苏农业学报,2011,27(5):1 141-1 147.ZHAO T M,YU W G,ZHAO L P,et al.Research progress in breeding of tomato resistance to Botrytis cinerea[J].Jiangsu Journal of Agricultural Sciences,2011,27(5):1 141-1 147.
[17]VEGA A,CANESSA P,HOPPE G,et al.Transcriptome analysis reveals regulatory networks underlying differential susceptibility to Botrytis cinerea in response to nitrogen availability in Solanum lycopersicum[J].Frontiers in Plant Science,2015,6(142):911-921.
[18]AUDENAERT K,PATTERY T,CORNELIS P,et al.Induction of systemic resistance to Botrytis cinerea in tomato by Pseudomonas aeruginosa 7NSK2:role of salicylic acid,pyochelin,and pyocyanin[J].Moleculer Plant-Microbe Interactions,2002,15(11):1 147-1 156.
[19]BLANCO-ULATE B,VINCENTI E,POWELL A L,et al.Tomato transcriptome and mutant analyses suggest a role for plant stress hormones in the interaction between fruit and Botrytis cinerea[J].Frontiers in Plant Science,2013,4(7):142-152.
[20]CANTU D,VICENTE A R,GREVE L C,et al.The intersection between cell wall disassembly,ripening,and fruit susceptibility to Botrytis cinerea[J].Proceedings of the National.Academy of Sciences of the United States of American,2008,105(3):859-864.
[21]JIN W,WU F.Characterization of miRNAs associated with Botrytis cinerea infection of tomato leaves[J].BMC Plant Biology,2015,15(15):1-14.
[22]李保聚,朱国仁,赵奎华,等.番茄灰霉病在果实上的侵染部位及防治新技术[J].植物病理学报,1999,1(12):63-67.LI B J,ZHU G R,ZHAO K H,et al.Infection gates of gery mould of tomato fruits and a new technique for its controlling[J].Acta Phytopathologica Sinica,1999,1(12):63-67.
[23]刘小花,张岚岚,朱长青,等.Micro-Tom番茄矮化微型机制及其在植物功能基因组学研究中的应用[J].遗传,2008,30(10):1 257-1 264.LIU X H,ZHANG L L,ZHU C Q,et al.Mechanisms for miniature dwarf characteristics of Micro-Tom tomato and its application in plant functional genomics studies[J].Hereditas,2008,30(10):1 257-1 264.
[24]余朝阁,魏岚,李天来,等.钙对水杨酸诱导番茄叶片灰霉病抗性及防御酶活性的影响[J].西北植物学报,2012,32(6):1 166-1 170.YU C G,WEI L,LI T L,et al.Effects of calcium on resistance to gray mold and aactivities of defensive eenzymes induced by salicylic acid in tomato leave[J].Acta Botanica Boreal-Occidentalia Sinica,2012,32(6):1 166-1 170.
[25]李琳琳,李天来,余朝阁,等.钙素对SA诱导番茄幼苗抗灰霉病的调控作用[J].园艺学报,2012,39(2):273-280.LI L L,LI T L,YU C G,et al.Effect of calcium on regulation of SA-induced Resistance to Botrytis cinerea in tomato[J].Acta Horticulturae Sinica,2012,39(2):273-280.
[26]LU M,WANG L F,DU X H,et al.Molecular cloning and expression analysis of jasmonic acid dependent but salicylic acid independent LeWRKY1[J].Genetics and Molecular Research,2015,14(4):15 390-15 398.
[27]ZHOU J,WANG J,ZHENG Z,et al.Characterization of the promoter and extended C-terminal domain of Arabidopsis WRKY33and functional analysis of tomato WRKY33homologues in plant stress responses[J].Journal of Experimental Botany,2015,66(15):4 567-4 583.
[28]ATAMIAN H S,EULGEM T,KALOSHIAN I.SlWRKY70is required for Mi-1-mediated resistance to aphids and nematodes in tomato[J].Planta,2012,235(2):299-309.
[29]HUANG W,MIAO M,KUD J,et al.SlNAC1,a stress-related transcription factor,is fine-tuned on both the transcriptional and the post-translational level[J].New Phytologist,2013,197(4):1 214-1 224.
[30]ZHU M,CHEN G,et al.The abiotic stress-responsive NACtype transcription factor SlNAC4regulates salt and drought tolerance and stress-related genes in tomato(Solanum lycopersicum)[J].Plant Cell Reports,2014,33(11):1 851-1 863.
[31]刘茜,王爱云,荣玮,等.ERF转录因子在作物抗病基因工程中的研究进展[J].种子,2014,33(1):48-52.LIU Q,WANG A Y,RONG W,et al.Progress of ERF transcription factors in genetic engineering for disease resistance in crops[J].Seed,2014,33(1):48-52.
[32]LIU M,GOMES B L,et al.Comprehensive profiling of ethylene response factor expression identifies ripening-associated ERF genes and their link to key regulators of fruit ripening in tomato[J].Plant Physiology,2016,170(3):1 732-1 744.
[33]KIM K N.Stress responses mediated by the CBL calcium sensors in plants[J].Plant Biotechnology Reports,2013,7(1):1-8.
[34]董连红,史素娟,苏玉龙,等.植物CBL基因家族的研究进展[J].核农学报,2015,9(5):892-898.DONG L H,SHI S J,SU Y L,et al.Advances in research of CBL family in plant[J].Journal of Nuclear Agricultural Sciences,2015,9(5):892-898.
[35]刘淑梅,王施慧,刘明毓,等.番茄CBL家族基因的鉴定和遗传进化分析[J].分子植物育种,2015,13(10):2 268-2 273.LIU S M,WANG S H,LIU M Y,et al.Identification and characterization of CBL family genes in tomato[J].Molecular Plant Breeding,2015,13(10):2 268-2 273.
[2]MARTINEZ-ATIENZA J,JIANG X,GARCIADEBLAS B,et al.Conservation of the salt overly sensitive pathway in rice[J].Plant Physiology,2007,143(2):1 001-1 012.
[3]WANG M Y,GU D,LIU T S,et al.Overexpression of a putative maize calcineurin B-like protein in Arabidopsis confers salt tolerance[J].Plant Molecular Biology,2007,65(6):733-746.
[4]SHI S S,CHEN W,SUNW N.Comparative proteomic analysis of the Arabidopsis cbl1 mutant in response to salt stress[J].Proteomics,2011,11(24):4 712-4 725.
[5]ALBRECHT V,WEINL S,BLAZEVIC D,et al.The calcium sensor CBL1intergrates plant responses to abiotic stresses[J].The Plant Journal,2003,36(4):457-470.
[6]CHEONG Y H,KIM K N,et al.CBL1,a calcium sensor that differentially regulates salt,drought,and cold responses in Arabidopsis[J].The Plant Cell,2003,15(8):1 833-1 845.
[7]马齐军,胡大刚,由春香,等.苹果MdCBL家族基因表达分析及MdCBL1的功能鉴定[J].园艺学报2014,41(6):1 053-1 062.MA Q J,HU D G,YOU C X,et al.Classification and expression analysis of apple MdCBLfamily genes with a functional characterization of MdCBL1[J].Acta Horticulturae Sinica,2014,41(6):1 053-1 062.
[8]韩金龙,李慧,丛郁,等.杜梨CBL1和CBL7基因对非生物逆境的响应[J].果树学报,2014,31(4):529-535.HAN J L,LI H,CONG Y,et al.Comparison of two CBL genes on stress tolerance functions from Pyrus betulaefolia[J].Journal of Fruit Science,2014,31(4):529-535.
[9]许园园,蔺经,李晓刚,等.梨CBL基因家族全基因组序列的鉴定及非生物胁迫下的表达分析[J].中国农业科学,2015,48(4):735-747.XU Y Y,LIN J,LI X G,et al.Identification and expression analysis under abiotic stresses of the CBL gene family in pear[J].Scientia Agricultura Sinica,2015,48(4):735-747.
[10]GAO P,ZHAO P M,WANG J,et al.Co-expression and preferential interaction between two calcineurin B-like proteins and a CBL-interacting protein kinase from cotton[J].Plant Physiology and Biochemistry,2008,46(10):935-940.
[11]赵晋锋,余爱丽,田岗,等.谷子CBL基因鉴定及其在干旱、高盐胁迫下的表达分析[J].作物学报,2013,39(2):360-367.ZHAO J F,YU A L,TIAN G,et al.Identification of CBL genes from foxtail millet(Setariaitalic[L.]Beauv.)and its expression under drought and salt stresses[J].Acta Agronomica Sinica,2013,39(2):360-367.
[12]ZHAO J F,SUN Z F,ZHENG J,et al.Cloning and characterization of a novel CBL-interacting protein kinase from maize[J].Plant Molecular Biology,2009,69(6):661-674.
[13]TOKAS I,PANDEY A,et al.Role of calcium-mediated CBLCIPK network in plant mineral nutrition and abiotic stress[M].Molecular Stress Physiology of Plants,2013:241-261.
[14]DRERUP M M,SCHLUCKING K,HASHIMOTO K,et al.The calcineurin B-like calcium sensors CBL1and CBL9together with their interacting protein kinase CIPK26regulate the Arabidopsis NADPH oxidase RBOHF[J].Molecular Plant,2013,6(2):559-569.
[15]MAHS A,STEINHORST L,HAN J P,et al.The calcineurin B-like Ca2+sensors CBL1and CBL9function in pollen germination and pollen tube growth in Arabidopsis[J].Molecular Plant,2013,6(4):1 149-1 162.
[16]赵统敏,余文贵,赵丽萍,等.番茄抗灰霉病育种研究进展[J].江苏农业学报,2011,27(5):1 141-1 147.ZHAO T M,YU W G,ZHAO L P,et al.Research progress in breeding of tomato resistance to Botrytis cinerea[J].Jiangsu Journal of Agricultural Sciences,2011,27(5):1 141-1 147.
[17]VEGA A,CANESSA P,HOPPE G,et al.Transcriptome analysis reveals regulatory networks underlying differential susceptibility to Botrytis cinerea in response to nitrogen availability in Solanum lycopersicum[J].Frontiers in Plant Science,2015,6(142):911-921.
[18]AUDENAERT K,PATTERY T,CORNELIS P,et al.Induction of systemic resistance to Botrytis cinerea in tomato by Pseudomonas aeruginosa 7NSK2:role of salicylic acid,pyochelin,and pyocyanin[J].Moleculer Plant-Microbe Interactions,2002,15(11):1 147-1 156.
[19]BLANCO-ULATE B,VINCENTI E,POWELL A L,et al.Tomato transcriptome and mutant analyses suggest a role for plant stress hormones in the interaction between fruit and Botrytis cinerea[J].Frontiers in Plant Science,2013,4(7):142-152.
[20]CANTU D,VICENTE A R,GREVE L C,et al.The intersection between cell wall disassembly,ripening,and fruit susceptibility to Botrytis cinerea[J].Proceedings of the National.Academy of Sciences of the United States of American,2008,105(3):859-864.
[21]JIN W,WU F.Characterization of miRNAs associated with Botrytis cinerea infection of tomato leaves[J].BMC Plant Biology,2015,15(15):1-14.
[22]李保聚,朱国仁,赵奎华,等.番茄灰霉病在果实上的侵染部位及防治新技术[J].植物病理学报,1999,1(12):63-67.LI B J,ZHU G R,ZHAO K H,et al.Infection gates of gery mould of tomato fruits and a new technique for its controlling[J].Acta Phytopathologica Sinica,1999,1(12):63-67.
[23]刘小花,张岚岚,朱长青,等.Micro-Tom番茄矮化微型机制及其在植物功能基因组学研究中的应用[J].遗传,2008,30(10):1 257-1 264.LIU X H,ZHANG L L,ZHU C Q,et al.Mechanisms for miniature dwarf characteristics of Micro-Tom tomato and its application in plant functional genomics studies[J].Hereditas,2008,30(10):1 257-1 264.
[24]余朝阁,魏岚,李天来,等.钙对水杨酸诱导番茄叶片灰霉病抗性及防御酶活性的影响[J].西北植物学报,2012,32(6):1 166-1 170.YU C G,WEI L,LI T L,et al.Effects of calcium on resistance to gray mold and aactivities of defensive eenzymes induced by salicylic acid in tomato leave[J].Acta Botanica Boreal-Occidentalia Sinica,2012,32(6):1 166-1 170.
[25]李琳琳,李天来,余朝阁,等.钙素对SA诱导番茄幼苗抗灰霉病的调控作用[J].园艺学报,2012,39(2):273-280.LI L L,LI T L,YU C G,et al.Effect of calcium on regulation of SA-induced Resistance to Botrytis cinerea in tomato[J].Acta Horticulturae Sinica,2012,39(2):273-280.
[26]LU M,WANG L F,DU X H,et al.Molecular cloning and expression analysis of jasmonic acid dependent but salicylic acid independent LeWRKY1[J].Genetics and Molecular Research,2015,14(4):15 390-15 398.
[27]ZHOU J,WANG J,ZHENG Z,et al.Characterization of the promoter and extended C-terminal domain of Arabidopsis WRKY33and functional analysis of tomato WRKY33homologues in plant stress responses[J].Journal of Experimental Botany,2015,66(15):4 567-4 583.
[28]ATAMIAN H S,EULGEM T,KALOSHIAN I.SlWRKY70is required for Mi-1-mediated resistance to aphids and nematodes in tomato[J].Planta,2012,235(2):299-309.
[29]HUANG W,MIAO M,KUD J,et al.SlNAC1,a stress-related transcription factor,is fine-tuned on both the transcriptional and the post-translational level[J].New Phytologist,2013,197(4):1 214-1 224.
[30]ZHU M,CHEN G,et al.The abiotic stress-responsive NACtype transcription factor SlNAC4regulates salt and drought tolerance and stress-related genes in tomato(Solanum lycopersicum)[J].Plant Cell Reports,2014,33(11):1 851-1 863.
[31]刘茜,王爱云,荣玮,等.ERF转录因子在作物抗病基因工程中的研究进展[J].种子,2014,33(1):48-52.LIU Q,WANG A Y,RONG W,et al.Progress of ERF transcription factors in genetic engineering for disease resistance in crops[J].Seed,2014,33(1):48-52.
[32]LIU M,GOMES B L,et al.Comprehensive profiling of ethylene response factor expression identifies ripening-associated ERF genes and their link to key regulators of fruit ripening in tomato[J].Plant Physiology,2016,170(3):1 732-1 744.
[33]KIM K N.Stress responses mediated by the CBL calcium sensors in plants[J].Plant Biotechnology Reports,2013,7(1):1-8.
[34]董连红,史素娟,苏玉龙,等.植物CBL基因家族的研究进展[J].核农学报,2015,9(5):892-898.DONG L H,SHI S J,SU Y L,et al.Advances in research of CBL family in plant[J].Journal of Nuclear Agricultural Sciences,2015,9(5):892-898.
[35]刘淑梅,王施慧,刘明毓,等.番茄CBL家族基因的鉴定和遗传进化分析[J].分子植物育种,2015,13(10):2 268-2 273.LIU S M,WANG S H,LIU M Y,et al.Identification and characterization of CBL family genes in tomato[J].Molecular Plant Breeding,2015,13(10):2 268-2 273.