‘云香’水仙NtNAC2基因的克隆及功能分析
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摘要
该研究选用多花水仙新品种‘云香’为材料,采用RT-PCR技术克隆得到NtNAC2,其开放阅读框为936bp,编码311个氨基酸。生物信息学分析显示,NtNAC2在N端含有NAM保守结构域,与单子叶植物芦笋、番红花和石斛等具有较近的进化关系。实时荧光定量检测显示,花瓣和副冠中NtNAC2基因的表达量随花器官发育逐渐上升,衰败期达到峰值;在ABA、MeJA、SA、H_2O_2、50℃、NaCl和PEG胁迫处理下,水仙根和叶中NtNAC2基因的表达量上调,且表现出时空表达特异性。为进一步鉴定其功能,构建植物表达载体转化烟草,获得10株转基因烟草,经半定量RT-PCR检测NtNAC2在转基因植株中过表达。高盐和干旱胁迫处理后转基因植株根长为野生型的2～3倍,叶片失水率低于30%,存活率高于60%。研究表明,‘云香’水仙NtNAC2的过量表达提高了转基因烟草的抗旱性和耐盐性,可作为水仙抗逆分子育种的重要候选基因。
In this study,we used the new species Narcissus tazetta 'Yunxiang'as the experimental material.A NAC gene was cloned by RT-PCR and named NtNAC2,which contains 936 bp open reading frame encoding 311 amino acids.Bioinformatics analysis showed that NtNAC2 gene is a member of NAC family,which contains a typical NAM conserved domain located in the N-terminal region and a close evolutionary relationship grouped with monocotyledonous Asparagus officinalis,Crocus sativus,Dendrobium catenatum.The quantitative real-time PCR detection showed that the relative expression level of NtNAC2 gene in petal and corona increased gradually with the development of floral organs,and reached the peak in the senescence period.The expression profiles of NtNAC2 in root and leaf were analyzed,it was inducible under ABA,MeJA,SA,H_2 O_2,50 ℃,PEG and NaCl treatments,and the spatio-temporal and tissue expression difference of root and leaf was reflected.In order to further identify its abiotic stress function,we successfully constructed the plant over-expression vector and finally obtained 10 strains of transgenic tobacco by GUS and molecular detection.The over-expression of NtNAC2 in transgenic tobacco was detected by semi-quantitative RT-PCR.The roots of transgenic plants are 2 to 3 times that of wild type,thewater loss rate of leaves is less than 30% and the survival rate is higher than 60% under the salt and drought stress.These results showed that the expression of NtNAC2 gene can increase the tolerance of tobacco to high salt and drought,so it can be used as an effective candidate gene for N.tazettaresistance molecular breeding.
In this study,we used the new species Narcissus tazetta 'Yunxiang'as the experimental material.A NAC gene was cloned by RT-PCR and named NtNAC2,which contains 936 bp open reading frame encoding 311 amino acids.Bioinformatics analysis showed that NtNAC2 gene is a member of NAC family,which contains a typical NAM conserved domain located in the N-terminal region and a close evolutionary relationship grouped with monocotyledonous Asparagus officinalis,Crocus sativus,Dendrobium catenatum.The quantitative real-time PCR detection showed that the relative expression level of NtNAC2 gene in petal and corona increased gradually with the development of floral organs,and reached the peak in the senescence period.The expression profiles of NtNAC2 in root and leaf were analyzed,it was inducible under ABA,MeJA,SA,H_2 O_2,50 ℃,PEG and NaCl treatments,and the spatio-temporal and tissue expression difference of root and leaf was reflected.In order to further identify its abiotic stress function,we successfully constructed the plant over-expression vector and finally obtained 10 strains of transgenic tobacco by GUS and molecular detection.The over-expression of NtNAC2 in transgenic tobacco was detected by semi-quantitative RT-PCR.The roots of transgenic plants are 2 to 3 times that of wild type,thewater loss rate of leaves is less than 30% and the survival rate is higher than 60% under the salt and drought stress.These results showed that the expression of NtNAC2 gene can increase the tolerance of tobacco to high salt and drought,so it can be used as an effective candidate gene for N.tazettaresistance molecular breeding.
引文
[1]NAKASHIMA K,TAKASAKAI H,MIZOI J,et al.NAC transcription factors in plant abiotic stress responses[J].Biochim Biophys Acta.2012,1 819(2):97-103.
[2]NURUZZAMAN H,SHARONI A M,KIKUCHI S.Roles of NAC transcription factors in the regulation of biotic and abiotic stress responses in plants[J].Frontiers in Microbiology,2013,4:248.
[3]UDVARDI M K,KAKAR K,WANDREY M,et al.Legume transcription factors:global regulators of plant development and response to the environment[J].Plant Physiology,2007,144(2):538-549.
[4]RUSHTON P J,BOKOWIEC M T,HAN S,et al.Tobacco transcription factors:novel insights into transcriptional regulation in the solanaceae[J].Plant Physiology,2008,147(1):280-295.
[5]NURUZZAMAN M,MANIMEKALAI R,SHARONI A M,et al.Genome-wide analysis of NAC transcription factor family in rice[J].Gene,2010,465(1-2):30-44.
[6]LE D T,NISHIYAMA R,WATANABE Y,et al.Genomewide survey and expression analysis of the plant-specific NACtranscription factor family in soybean during development and dehydration stress[J].DNA Research,2011,18(4):263-276.
[7]PURANIK S,SAHU P P,SRIVASTAVA P S,et al.NAC proteins:regulation and role in stress tolerance[J].Trends in Plant Science,2012,17(6):369-381.
[8]OOKA H,SATOH K,DOI K,et al.Comprehensive analysis of NAC family genes in Oryza sativa and Arabidopsis thaliana[J].DNA Research,2003,10(6):239-247.
[9]WU Y R,DENG Z Y,LAI J B,et al.Dual function of Arabidopsis ATAF1in abiotic and biotic stress responses[J].Cell Research,2009,19(11):1 279-1 290.
[10]HU H,DAI M,YAO J,et al.Overexpressing a NAM,AT-AF,and CUC(NAC)transcription factor enhances drought resistance and salt tolerance in rice[J].Proceedings of the National Academy of Sciences of the United States of A-merica,2006,103(35):12 987-12 992.
[11]SAAD A S,LI X,LI H P,et al.A rice stress-responsive NACgene enhances tolerance of transgenic wheat to drought and salt stresses[J].Plant Science,2013,203-204(35):33-40.
[12]LIU G Z,LI X L,JIN S X,et al.Overexpression of rice NACGene SNAC1improves drought and salt tolerance by enhancing root development and reducing transpiration rate in transgenic cotton[J].Plos One,2014,9(1):e868951.
[13]HUANG Q J,WANG Y,LI B,et al.TaNAC29,a NAC transcription factor from wheat,enhances salt and drought tolerance in transgenic Arabidopsis[J].BMC Plant Biology,2015,15:268.
[14]LIU Y M,YU X W,LIU S S,et al.A chickpea NAC-type transcription factor,CarNAC6,confers enhanced dehydration tolerance in Arabidopsis[J].Plant Molecular Biology Reporter,2017,35(1):83-96.
[15]CHEN D,CHAI S,MCINTYRE C L,et al.Overexpression of apredominantly root-expressed NAC transcription factor in wheat roots enhances root length,biomass and drought tolerance[J].Plant Cell Reporter,2018,37(2):225-237.
[16]DAI F,ZHANG C Q,JIANG X Q,et al.RhNAC2and RhEX-PA4are involved in the regulation of dehydration tolerance during the expansion of rose petals[J].Plant Physiology,2012,160(4):2 064-2 082.
[17]JIANG X Q,ZHANG C Q,LU P T,et al.RhNAC3,a stressassociated NAC transcription factor,has a role in dehydration tolerance through regulating osmotic stress-related genes in rose petals[J].Plant Biotechnology Journal,2014,12(1):38-48.
[18]NEGI S,TAK H,GANAPATHI T R.A banana NAC transcription factor(MusaSNAC1)impart drought tolerance by modulating stomatal closure and H2O2content[J].Plant Molecular Biology,2018,96(4-5):457-471.
[19]CAO H S,WANG L,NAWAZ M A,et al.Ectopic expression of pumpkin NAC transcription factor CmNAC1improves multiple abiotic stress tolerance in Arabidopsis[J].Frontiers in Plant Science,2017,8:2 052.
[20]LU X,ZHANG X,DUAN H,et al.Three stress-responsive NAC transcription factors fromPopulus euphratica differentially regulate salt and drought tolerance in transgenic plants[J].Physiologia Plantarum,2018,162(1):73-97.
[21]李梦思,赵潇俐,吴用,等.‘云香’水仙NAC基因克隆及表达分析[J].热带作物学报,2017,38(2):313-319.LI M S,ZHAO X L,WU Y,et al.Cloning and expression analysis of NAC gene from Narcissus tazetta var.chinesis‘Yunxiang’[J].Chinese Journal of Tropical Crops,2017,38(2):313-319.
[22]陈晓静.福建3个产地水仙的核型分析.植物资源与环境学报[J],2004,13(4):28-31.CHEN X J.Analysis of karyotypes of three localities of Narcissus tazetta var.chinensis in Fujian Province[J].Journal of Plant Resources and Environment,2004,13(4):28-31.
[23]温秀萍,孙申申,杨菲颖,等.‘云香’水仙NtWRKYY2基因的克隆及功能分析[J].西北植物学报,2017,37(7):1 255-1 262.WEN X P,SUN S S,YANG F Y,et al.Cloning and function analysis of NtWRKYY2from Narcissus tazetta var.‘Yunxiang’[J].Acta Botanica Boreali-Occidentalia Sinica,2017,37(17):1 255-1 262.
[24]孙申申,温秀萍,杨菲颖,等.‘云香’水仙ACC合成酶基因NtACS1的克隆及遗传转化[J].西北植物学报,2017,37(2):250-257.SUN S S,WEN X P,YANG F Y,et al.Molecular cloning and its transformation of ACC synthase NtACSl gene from Narcissus tazetta var.‘Yunxiang’[J].Acta Botanica Boreali-Occidentalia Sinica,2017,37(2):250-257.
[25]律凤霞.根癌农杆菌介导外源基因转化烟草体系的优化[J].安徽农业科学,2010,38(19):10 065-10 066.LUF X.Optimization for agrobacterium tumefaciens-mediated transformation system for exogenous genes of tobacco[J].Journal of Anhui Agricultural Sciences,2010,38(19):10 065-10 066.
[26]TRAN L,NAKASHIMA K,SAKUMA Y,et al.Isolation and functional analysis of Arabidopsis stress-inducible NAC transcription factors that bind to a drought-responsive cis-element in the early responsive to dehydration stress 1promoter[J].Plant Cell,2004,16(9):2 481-2 498.
[27]SHAO H B,WANG H Y,TANG X L.NAC transcription factors in plant multiple abiotic stress responses:progress and prospects[J].Frontiers in Plant Science,2015,6:902.
[28]HE X J,MU R L,CAO W H,et al.AtNAC2,a transcription factor downstream of ethylene and auxin signaling pathways,is involved in salt stress response and lateral root development[J].Plant Journal,2010,44(6):903-916.
[29]XU Z Y,WANG C Y,XUE F,et al.Wheat NAC transcription factor TaNAC29is involved in response to salt stress[J].Plant Physiology&Biochemistry,2015,96:356-363.
[30]申玉华,徐振军,唐立红,等.紫花苜蓿NAC类转录因子基因MsNAC2的克隆及其功能分析[J].中国农业科学,2015,48(15):2 925-2 938.SHEN Y H,XU Z J,TANG L H,et al.Cloning and function analysis of the MsNAC2gene with NAC transcription factor from alfalfa[J].Scientia Agricultura Sinica,2015,48(15):2 925-2 938.
[31]申玉华,李雪梅,乔晓慧,等.紫花苜蓿逆境响应转录因子MsNAC3基因克隆及表达分析[J].西北植物学报,2017,37(10):1 919-1 925.SHEN Y H,LI X M,QIAO X H,et al.Cloning and expression of the NAC transcription factor gene MsNAC3gene with NAC transcription factor from Medicago sativa L.[J].Acta Botanica Boreali-Occidentalia Sinica,2017,37(10):1 919-1 925.
[32]HAO Y J,WEI W,SONG Q X,et al.Soybean NAC transcription factors promote abiotic stress tolerance and lateral root formation in transgenic plants[J].Plant Journal,2011,68(2):302-313.
[33]杨再强,邱译萱,刘朝霞,等.土壤水分胁迫对设施番茄根系及地上部生长的影响[J].生态学报,2016,36(3):748-757.YANG Z Q,QIU Y X,LIU Z X,et al.The effects of soil moisture stress on the growth of root and above-ground parts of greenhouse tomato crops[J].Chinese Journal of Ecology,2016,36(3):748-757.
[34]ZHENG X N,CHEN B,LU G J,et al.Overexpression of a NAC transcription factor enhances rice drought and salt tolerance[J].Biochemical and Biophysical Research Communications,2009,379(4):985-989.
[35]YOKOTANI N,ICHIKAWA T,KONDOU Y,et al.Tolerance to various environmental stresses conferred by the saltresponsive rice gene ONAC063in transgenic Arabidopsis[J].Planta,2009,229(5):1 065-1 075.
[36]ZHANG L N,ZHANG L C,XIA C,et al.The novel wheat transcription factor TaNAC47 enhances multiple abiotic stress tolerances in transgenic plants[J].Frontiers in Plant Science,2016,6:1 174.
[37]LIU Z J,LI F,WANG L G,et al.Molecular characterization of a stress-induced NAC gene,GhSNAC3,from Gossypium hirsutum[J].Journal of Genetics,2018,97(2):1-10.
[38]XU X Y,YAO X Z,LU L T,et al.Overexpression of the transcription factor NtNAC2,confers drought tolerance in tobacco[J].Plant Molecular Biology Reporter,2018,36(12):1-10.
[39]MAO X G,ZHANG H Y,QIAN X Y,et al.TaNAC2,a NAC-type wheat transcription factor conferring enhanced multiple abiotic stress tolerances in Arabidopsis[J].Journal of Experimental Botany,2012,63(8):2 933-2 946.
[40]GUO Y N,PANG C Y,JIA X Y,et al.An NAM domain gene,GhNAC79,improves resistance to drought stress in upland cotton[J].Frontiers in Plant Science,2017,8:1 657.
[2]NURUZZAMAN H,SHARONI A M,KIKUCHI S.Roles of NAC transcription factors in the regulation of biotic and abiotic stress responses in plants[J].Frontiers in Microbiology,2013,4:248.
[3]UDVARDI M K,KAKAR K,WANDREY M,et al.Legume transcription factors:global regulators of plant development and response to the environment[J].Plant Physiology,2007,144(2):538-549.
[4]RUSHTON P J,BOKOWIEC M T,HAN S,et al.Tobacco transcription factors:novel insights into transcriptional regulation in the solanaceae[J].Plant Physiology,2008,147(1):280-295.
[5]NURUZZAMAN M,MANIMEKALAI R,SHARONI A M,et al.Genome-wide analysis of NAC transcription factor family in rice[J].Gene,2010,465(1-2):30-44.
[6]LE D T,NISHIYAMA R,WATANABE Y,et al.Genomewide survey and expression analysis of the plant-specific NACtranscription factor family in soybean during development and dehydration stress[J].DNA Research,2011,18(4):263-276.
[7]PURANIK S,SAHU P P,SRIVASTAVA P S,et al.NAC proteins:regulation and role in stress tolerance[J].Trends in Plant Science,2012,17(6):369-381.
[8]OOKA H,SATOH K,DOI K,et al.Comprehensive analysis of NAC family genes in Oryza sativa and Arabidopsis thaliana[J].DNA Research,2003,10(6):239-247.
[9]WU Y R,DENG Z Y,LAI J B,et al.Dual function of Arabidopsis ATAF1in abiotic and biotic stress responses[J].Cell Research,2009,19(11):1 279-1 290.
[10]HU H,DAI M,YAO J,et al.Overexpressing a NAM,AT-AF,and CUC(NAC)transcription factor enhances drought resistance and salt tolerance in rice[J].Proceedings of the National Academy of Sciences of the United States of A-merica,2006,103(35):12 987-12 992.
[11]SAAD A S,LI X,LI H P,et al.A rice stress-responsive NACgene enhances tolerance of transgenic wheat to drought and salt stresses[J].Plant Science,2013,203-204(35):33-40.
[12]LIU G Z,LI X L,JIN S X,et al.Overexpression of rice NACGene SNAC1improves drought and salt tolerance by enhancing root development and reducing transpiration rate in transgenic cotton[J].Plos One,2014,9(1):e868951.
[13]HUANG Q J,WANG Y,LI B,et al.TaNAC29,a NAC transcription factor from wheat,enhances salt and drought tolerance in transgenic Arabidopsis[J].BMC Plant Biology,2015,15:268.
[14]LIU Y M,YU X W,LIU S S,et al.A chickpea NAC-type transcription factor,CarNAC6,confers enhanced dehydration tolerance in Arabidopsis[J].Plant Molecular Biology Reporter,2017,35(1):83-96.
[15]CHEN D,CHAI S,MCINTYRE C L,et al.Overexpression of apredominantly root-expressed NAC transcription factor in wheat roots enhances root length,biomass and drought tolerance[J].Plant Cell Reporter,2018,37(2):225-237.
[16]DAI F,ZHANG C Q,JIANG X Q,et al.RhNAC2and RhEX-PA4are involved in the regulation of dehydration tolerance during the expansion of rose petals[J].Plant Physiology,2012,160(4):2 064-2 082.
[17]JIANG X Q,ZHANG C Q,LU P T,et al.RhNAC3,a stressassociated NAC transcription factor,has a role in dehydration tolerance through regulating osmotic stress-related genes in rose petals[J].Plant Biotechnology Journal,2014,12(1):38-48.
[18]NEGI S,TAK H,GANAPATHI T R.A banana NAC transcription factor(MusaSNAC1)impart drought tolerance by modulating stomatal closure and H2O2content[J].Plant Molecular Biology,2018,96(4-5):457-471.
[19]CAO H S,WANG L,NAWAZ M A,et al.Ectopic expression of pumpkin NAC transcription factor CmNAC1improves multiple abiotic stress tolerance in Arabidopsis[J].Frontiers in Plant Science,2017,8:2 052.
[20]LU X,ZHANG X,DUAN H,et al.Three stress-responsive NAC transcription factors fromPopulus euphratica differentially regulate salt and drought tolerance in transgenic plants[J].Physiologia Plantarum,2018,162(1):73-97.
[21]李梦思,赵潇俐,吴用,等.‘云香’水仙NAC基因克隆及表达分析[J].热带作物学报,2017,38(2):313-319.LI M S,ZHAO X L,WU Y,et al.Cloning and expression analysis of NAC gene from Narcissus tazetta var.chinesis‘Yunxiang’[J].Chinese Journal of Tropical Crops,2017,38(2):313-319.
[22]陈晓静.福建3个产地水仙的核型分析.植物资源与环境学报[J],2004,13(4):28-31.CHEN X J.Analysis of karyotypes of three localities of Narcissus tazetta var.chinensis in Fujian Province[J].Journal of Plant Resources and Environment,2004,13(4):28-31.
[23]温秀萍,孙申申,杨菲颖,等.‘云香’水仙NtWRKYY2基因的克隆及功能分析[J].西北植物学报,2017,37(7):1 255-1 262.WEN X P,SUN S S,YANG F Y,et al.Cloning and function analysis of NtWRKYY2from Narcissus tazetta var.‘Yunxiang’[J].Acta Botanica Boreali-Occidentalia Sinica,2017,37(17):1 255-1 262.
[24]孙申申,温秀萍,杨菲颖,等.‘云香’水仙ACC合成酶基因NtACS1的克隆及遗传转化[J].西北植物学报,2017,37(2):250-257.SUN S S,WEN X P,YANG F Y,et al.Molecular cloning and its transformation of ACC synthase NtACSl gene from Narcissus tazetta var.‘Yunxiang’[J].Acta Botanica Boreali-Occidentalia Sinica,2017,37(2):250-257.
[25]律凤霞.根癌农杆菌介导外源基因转化烟草体系的优化[J].安徽农业科学,2010,38(19):10 065-10 066.LUF X.Optimization for agrobacterium tumefaciens-mediated transformation system for exogenous genes of tobacco[J].Journal of Anhui Agricultural Sciences,2010,38(19):10 065-10 066.
[26]TRAN L,NAKASHIMA K,SAKUMA Y,et al.Isolation and functional analysis of Arabidopsis stress-inducible NAC transcription factors that bind to a drought-responsive cis-element in the early responsive to dehydration stress 1promoter[J].Plant Cell,2004,16(9):2 481-2 498.
[27]SHAO H B,WANG H Y,TANG X L.NAC transcription factors in plant multiple abiotic stress responses:progress and prospects[J].Frontiers in Plant Science,2015,6:902.
[28]HE X J,MU R L,CAO W H,et al.AtNAC2,a transcription factor downstream of ethylene and auxin signaling pathways,is involved in salt stress response and lateral root development[J].Plant Journal,2010,44(6):903-916.
[29]XU Z Y,WANG C Y,XUE F,et al.Wheat NAC transcription factor TaNAC29is involved in response to salt stress[J].Plant Physiology&Biochemistry,2015,96:356-363.
[30]申玉华,徐振军,唐立红,等.紫花苜蓿NAC类转录因子基因MsNAC2的克隆及其功能分析[J].中国农业科学,2015,48(15):2 925-2 938.SHEN Y H,XU Z J,TANG L H,et al.Cloning and function analysis of the MsNAC2gene with NAC transcription factor from alfalfa[J].Scientia Agricultura Sinica,2015,48(15):2 925-2 938.
[31]申玉华,李雪梅,乔晓慧,等.紫花苜蓿逆境响应转录因子MsNAC3基因克隆及表达分析[J].西北植物学报,2017,37(10):1 919-1 925.SHEN Y H,LI X M,QIAO X H,et al.Cloning and expression of the NAC transcription factor gene MsNAC3gene with NAC transcription factor from Medicago sativa L.[J].Acta Botanica Boreali-Occidentalia Sinica,2017,37(10):1 919-1 925.
[32]HAO Y J,WEI W,SONG Q X,et al.Soybean NAC transcription factors promote abiotic stress tolerance and lateral root formation in transgenic plants[J].Plant Journal,2011,68(2):302-313.
[33]杨再强,邱译萱,刘朝霞,等.土壤水分胁迫对设施番茄根系及地上部生长的影响[J].生态学报,2016,36(3):748-757.YANG Z Q,QIU Y X,LIU Z X,et al.The effects of soil moisture stress on the growth of root and above-ground parts of greenhouse tomato crops[J].Chinese Journal of Ecology,2016,36(3):748-757.
[34]ZHENG X N,CHEN B,LU G J,et al.Overexpression of a NAC transcription factor enhances rice drought and salt tolerance[J].Biochemical and Biophysical Research Communications,2009,379(4):985-989.
[35]YOKOTANI N,ICHIKAWA T,KONDOU Y,et al.Tolerance to various environmental stresses conferred by the saltresponsive rice gene ONAC063in transgenic Arabidopsis[J].Planta,2009,229(5):1 065-1 075.
[36]ZHANG L N,ZHANG L C,XIA C,et al.The novel wheat transcription factor TaNAC47 enhances multiple abiotic stress tolerances in transgenic plants[J].Frontiers in Plant Science,2016,6:1 174.
[37]LIU Z J,LI F,WANG L G,et al.Molecular characterization of a stress-induced NAC gene,GhSNAC3,from Gossypium hirsutum[J].Journal of Genetics,2018,97(2):1-10.
[38]XU X Y,YAO X Z,LU L T,et al.Overexpression of the transcription factor NtNAC2,confers drought tolerance in tobacco[J].Plant Molecular Biology Reporter,2018,36(12):1-10.
[39]MAO X G,ZHANG H Y,QIAN X Y,et al.TaNAC2,a NAC-type wheat transcription factor conferring enhanced multiple abiotic stress tolerances in Arabidopsis[J].Journal of Experimental Botany,2012,63(8):2 933-2 946.
[40]GUO Y N,PANG C Y,JIA X Y,et al.An NAM domain gene,GhNAC79,improves resistance to drought stress in upland cotton[J].Frontiers in Plant Science,2017,8:1 657.