刚毛柽柳NAC24基因的表达及抗逆功能分析
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摘要
【目的】NAC类转录因子是植物特有的最大的转录因子家族之一,广泛参与植物的生长发育过程,并在植物响应盐、干旱等多种非生物胁迫的过程中发挥至关重要的调控作用。本研究拟从盐生木本植物刚毛柽柳中克隆获得一个NAC转录因子基因,研究该基因的耐盐、抗旱功能,以期为研究木本植物NAC转录因子的抗逆分子机制奠定理论基础。【方法】在刚毛柽柳NaHCO_3胁迫转录组数据库中筛选获得一个NAC转录因子基因,将其命名为ThNAC24(GenBank登陆号:KF031949)。利用生物信息学工具将其与其他9个物种的NAC蛋白进行多序列比对,与拟南芥105个NAC蛋白进行进化树分析。分别用300 mmol·L~(-1) NaCl和400 mmol·L~(-1)甘露醇对刚毛柽柳进行胁迫,在胁迫6、12、24和48 h后分别取刚毛柽柳根及叶组织。通过实时荧光定量RT-PCR(qRT-PCR)技术分析盐、干旱胁迫下ThNAC24基因在不同胁迫时间点及不同组织的表达情况,初步鉴定其是否响应盐、干旱胁迫。为进一步研究ThNAC24基因的抗逆功能,分别构建植物过表达(pROKⅡ-ThNAC24)及抑制表达(pFGC5941-ThNAC24)载体。利用农杆菌介导的高效瞬时遗传转化体系获得ThNAC24基因瞬时过表达(OE)、抑制表达(IE)及对照(Control)刚毛柽柳植株。在盐、干旱胁迫下分析比较了ThNAC24基因瞬时过表达、抑制表达及对照刚毛柽柳植株的二氨基联苯胺(DAB)和氯化硝基四氮唑蓝(NBT)染色情况,过氧化物酶(POD)和超氧化物歧化酶(SOD)活性,及电解质渗透率、失水率及丙二醛(MDA)含量,鉴定ThNAC24基因的耐盐、抗旱功能。【结果】ThNAC24基因的开放阅读框为1 023 bp,编码340个氨基酸。多序列比对结果显示ThNAC24在N端的氨基酸序列相似度比较高,具有NAC家族的序列特征;系统进化树分析结果显示ThNAC24与ANAC103和ANAC082的亲缘关系较近。qRT-PCR结果显示:盐胁迫下,ThNAC24基因上调表达,在根组织中胁迫12 h表达量最高,而叶组织中胁迫24 h的表达量最高;干旱胁迫下,ThNAC24基因上调表达,在根组织中胁迫6 h表达量最高,在叶组织中胁迫12 h的表达量最高。ThNAC24基因在刚毛柽柳根和叶组织中均有表达且响应盐和干旱胁迫。过表达ThNAC24基因显著降低了刚毛柽柳H_2O_2和超氧阴离子含量,增强了POD和SOD酶的活性,从而减少活性氧(ROS)的积累。过表达ThNAC24基因能够降低刚毛柽柳在逆境胁迫下的电解质渗透率、失水率及MDA的积累,从而保护细胞膜结构的完整性。【结论】刚毛柽柳ThNAC24基因能够响应盐、干旱胁迫,过表达ThNAC24基因植株通过增强POD和SOD活性,进而提高ROS清除能力,减少细胞受损或死亡,从而提高刚毛柽柳的耐盐及抗旱能力。
【Objective】 The NAC transcription factors are one of the largest families of plant-specific transcription factors, widely involved in plant growth and development process, and it plays a crucial role in the process responding to salt, drought and many other abiotic stresses. In present study, we cloned a NAC transcription factors gene from halophyte woody plant Tamarix hispida, to study its salt tolerance and drought resistance function in order to lay a theoretical foundation for studying the molecular mechanism of resistance to woody plant NAC transcription factors.【Method】 A NAC transcription factor gene was isolated from NaHCO_3 stress transcriptome database of T. hispida, and it was named as ThNAC24(GenBank number: KF031949). Bioinformatics tools were used to perform multiple sequence alignment and phylogenetic analysis of NAC proteins from nine other species and 105 NAC proteins in Arabidopsis thaliana were analyzed. The T. hispida samples were treated with 300 mmol·L~(-1) NaCl and 400 mmol·L~(-1) Mannitol respectively, and the root and leaf tissues were taken at 6, 12, 24 and 48 h after the stress. The real-time fluorescent quantitative RT-PCR(qRT-PCR) was used to analyze the expression of ThNAC24 gene under salt or drought stress in different time and tissues. It was initially identified whether it responded to salt or drought stress. To further study the stress resistance function of ThNAC24, overexpression(pROKⅡ-ThNAC24) and inhibitory expression vectors(pFGC5941-ThNAC24) were constructed. Overexpression(OE), inhibitory expression(IE) and control T. hispida plants were obtained by Agrobacterium-mediated high-efficient transient transformation system. The DAB, NBT staining, POD activity, SOD activity, electrolyte leakage, water loss rate, and MDA content under salt and drought stress were measured and compared with the transgenic T. hispida to identify the salt tolerance and drought resistance of the ThNAC24 gene.【Result】 The open reading frame of ThNAC24 gene is 1 023 bp, encoding 340 amino acids. Multiple sequence alignments results show that the amino acid sequence of ThNAC24 is relatively high in the N-terminal and has the sequence characteristics of NAC family. Phylogenetic tree analysis showed that ThNAC24 was closely related to ANAC103 and ANAC082. The result of qRT-PCR showed that the expression of ThNAC24 gene was up-regulated under salt or drought stress. The expression level of ThNAC24 was the highest at 12 h in root tissue, while it was the highest at 24 h in leaf tissue; Under drought stress, the expression level was the highest at 6 h in root tissue, while it was the highest at 12 h in leaf tissue. The ThNAC24 gene was expressed in roots and leaves of T. hispida and responded to salt and drought stress. Overexpression of the ThNAC24 significantly reduced the contents of H_2O_2 and superoxide anion, enhanced the POD and SOD activities in T. hispida, thus decreased ROS accumulation. Overexpression of the ThNAC24 gene can reduce the electrolyte leakage, water loss rate and MDA accumulation of T. hispida under stress, thus protecting the integrity of cell membrane structure.【Conclusion】 The ThNAC24 gene can respond to salt and drought stress. Overexpression of the ThNAC24 gene significantly enhances POD and SOD activities to improve ROS scavenging, reduces cell damage or death of T. hispida and improves its salt tolerance and drought resistance.
【Objective】 The NAC transcription factors are one of the largest families of plant-specific transcription factors, widely involved in plant growth and development process, and it plays a crucial role in the process responding to salt, drought and many other abiotic stresses. In present study, we cloned a NAC transcription factors gene from halophyte woody plant Tamarix hispida, to study its salt tolerance and drought resistance function in order to lay a theoretical foundation for studying the molecular mechanism of resistance to woody plant NAC transcription factors.【Method】 A NAC transcription factor gene was isolated from NaHCO_3 stress transcriptome database of T. hispida, and it was named as ThNAC24(GenBank number: KF031949). Bioinformatics tools were used to perform multiple sequence alignment and phylogenetic analysis of NAC proteins from nine other species and 105 NAC proteins in Arabidopsis thaliana were analyzed. The T. hispida samples were treated with 300 mmol·L~(-1) NaCl and 400 mmol·L~(-1) Mannitol respectively, and the root and leaf tissues were taken at 6, 12, 24 and 48 h after the stress. The real-time fluorescent quantitative RT-PCR(qRT-PCR) was used to analyze the expression of ThNAC24 gene under salt or drought stress in different time and tissues. It was initially identified whether it responded to salt or drought stress. To further study the stress resistance function of ThNAC24, overexpression(pROKⅡ-ThNAC24) and inhibitory expression vectors(pFGC5941-ThNAC24) were constructed. Overexpression(OE), inhibitory expression(IE) and control T. hispida plants were obtained by Agrobacterium-mediated high-efficient transient transformation system. The DAB, NBT staining, POD activity, SOD activity, electrolyte leakage, water loss rate, and MDA content under salt and drought stress were measured and compared with the transgenic T. hispida to identify the salt tolerance and drought resistance of the ThNAC24 gene.【Result】 The open reading frame of ThNAC24 gene is 1 023 bp, encoding 340 amino acids. Multiple sequence alignments results show that the amino acid sequence of ThNAC24 is relatively high in the N-terminal and has the sequence characteristics of NAC family. Phylogenetic tree analysis showed that ThNAC24 was closely related to ANAC103 and ANAC082. The result of qRT-PCR showed that the expression of ThNAC24 gene was up-regulated under salt or drought stress. The expression level of ThNAC24 was the highest at 12 h in root tissue, while it was the highest at 24 h in leaf tissue; Under drought stress, the expression level was the highest at 6 h in root tissue, while it was the highest at 12 h in leaf tissue. The ThNAC24 gene was expressed in roots and leaves of T. hispida and responded to salt and drought stress. Overexpression of the ThNAC24 significantly reduced the contents of H_2O_2 and superoxide anion, enhanced the POD and SOD activities in T. hispida, thus decreased ROS accumulation. Overexpression of the ThNAC24 gene can reduce the electrolyte leakage, water loss rate and MDA accumulation of T. hispida under stress, thus protecting the integrity of cell membrane structure.【Conclusion】 The ThNAC24 gene can respond to salt and drought stress. Overexpression of the ThNAC24 gene significantly enhances POD and SOD activities to improve ROS scavenging, reduces cell damage or death of T. hispida and improves its salt tolerance and drought resistance.
引文
王关林,方宏筠.2016.植物基因工程实验技术指南.2版.北京:科学出版社,273-274,277-278.(Wang G L,Fang H J.2016.Laboratory guide for plant genetic engineering.2nd ed.Beijing:Science Press,273-274,277-278.[in Chinese])
王玉成,薄海侠,杨传平.2003.胡杨、柽柳总RNA提取方法的建立.东北林业大学学报,(5):99-100.(Wang Y C,Bo H Y,Yang C P.2003.A method for rapid isolation of total RNA from Tamarix and Populus euphratica Oliv.Journal of Northeast Forestry University,(5):99-100.[in Chinese])
杨勇,田新权,刘会利,等.2017.陆地棉NAC转录因子基因GhNAC6的克隆、表达和耐盐性分析.棉花学报,29(2):138-146.(Yang Y,Tian X Q,Liu H L,et al.2017.Cloning,expression and salt-tolerance analysis of the NAC transcription factor gene GhNAC6 in Upland Cotton.Cotton Science,29(2):138-146.[in Chinese])
尹林克.1995.中亚荒漠生态系统中的关键种—柽柳(Tamarix spp).干旱区研究,12(3):43-47.(Yin L K.1995.Tamarix spp.—The key species of desert ecosystem.Arid Zone Research,12(3):43-47.[in Chinese])
袁义杭,张鹤华,游韩莉,等.2018.青杄PwNAC42基因的克隆及表达模式分析.生物技术通报,34(4):1-6.(Yuan Y H,Zhang H H,You H L,et al.2018.Cloning and expression analysis of PwNAC42 in Picea wilsonii.Biotechnology Bulletin,34(4):1-6.[in Chinese])
Chen Q,Niu F,Yan J,et al.2017.Oilseed rape NAC56 transcription factor modulates reactive oxygen species accumulation and hypersensitive response-like cell death.Physiol Plant,160(2):209-221.
Duval M,Hsieh T F,Kim S Y,et al.2002.Molecular characterization of AtNAMa:member of the Arabidopsis NAC domain superfamily.Plant Molecular Biology,50(2):237-248.
Fang Y,Liao K,Du H,et al.2015.A stress-responsive NAC transcription factor SNAC3 confers heat and drought tolerance through modulation of reactive oxygen species in rice.J Exp Bot,66(21):6803-6817.
Hao Y J,Wei W,Song Q X,et al.2011.Soybean NAC transcription factors promote abiotic stress tolerance and lateral root formation in transgenic plants.The Plant Journal,68(2):302-313.
Hu H H,Dai M Q,Yao J L,et al.2006.Overexpressing a NAM,ATAF,and CUC (NAC) transcription factor enhances drought resistance and salt tolerance in rice.Proc Natl Acad Sci USA,103(35):12987-12992.
Huang D,Wang S,Zhang B,et al.2015.A gibberellin-mediated DELLA-NAC signaling cascade regulates cellulose synthesis in rice.Plant Cell,27(6):1681-1696.
Ji X Y,Zheng L,Liu Y J,et al.2014.A transient transformation system for the functional characterization of genes involved in stress response.Plant Mol Biol Rep,32(3):732-739.
Kjaersgaard T,Jensen M K,Christiansen M W,et al.2011.Senescence-associated barley NAC (NAM,ATAF1,2,CUC) transcription factor interacts with radical-induced cell death 1 through a disordered regulatory domain.Journal of Biological Chemistry,286(41):35418-35429.
Livak K J,Schmittgen T D.2001.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method.Methods,25(4):402-408.
Mao X,Zhang H,Qian X,et al.2012.TaNAC2,a NAC-type wheat transcription factor conferring enhanced multiple abiotic stress tolerances in Arabidopsis.Journal of Experimental Botany,63(8):2933-2946.
Nakashima K,Takasaki H,Mizoi J,et al.2012.NAC transcription factors in plant abiotic stress responses.Biochimica et Biophysica Acta-Gene Regulatory Mechanisma,1819(2):97-103.
Niu F,Wang C,Yan J,et al.2016.Functional characterization of NAC55 transcription factor from oilseed rape (Brassica napus L.) as a novel transcription alactivator modulating reactive oxygen species accumulation and cell death.Plant Mol Biol,92(1/2):89-104.
Ooka H,Satoh K,Doi K,et al.2003.Comprehensive analysis of NAC family genes in Oryza sativa and Arabidopsis thaliana.DNA Research,10(6):239-247.
Shen H,Yin Y,Chen F,et al.2009.A bioinformatic analysis of NAC genes for plant cell wall development in relation to lignocellulosic bioenergy production.Bioenergy Research,2(4):217-232.
Tang Y M,Liu M Y,Gao S Q,et al.2012.Molecular characterization of novel TaNAC genes in wheat and overexpression of TaNAC2a confers drought tolerance in tobacco.Physiol Plant,114(3):210-224.
Tran L S,Nakashima K,Sakuma Y,et al.2004.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 1 promoter.Plant Cell,16(9):2481-2498.
Tran L S,Nishiyama R,Yamaguchi-Shinozaki K,et al.2010.Potential utilization of NAC transcription factors to enhance abiotic stress tolerance in plants biotechnological approach.GM Crops,1(1):32-39.
Wang L,Li Z,Lu M,et al.2017b.ThNAC13,a NAC transcription factor from Tamarix hispida,confers salt and osmotic stress tolerance to transgenic Tamarix and Arabidopsis.Front Plant Sci,8:635.
Wang L L,Hu Z L,Zhu M K,et al.2017a.The abiotic stress-responsive NAC transcription factor SlNAC11 is involved in drought and salt response in tomato (Solanum lycopersicum L.).Plant Cell Tissue and Organ Culture,129(1):161-174.
Wang Z,Dane F.2013.NAC (NAM/ATAF/CUC) transcription factors in different stresses and their signaling pathway.Acta Physiologiae Plantarum,35(5):1397-1408.
Xia N,Zhang G,Liu X Y,et al.2010a.Characterization of a novel wheat NAC transcription factor gene involved in defense response against stripe rust pathogen infection and abiotic stresses.Molecular Biology Reports,37(8):3703-3712.
Xia N,Zhang G,Sun Y F,et al.2010b.TaNAC8,a novel NAC transcription factor gene in wheat,responds to stripe rust pathogen infection and abiotic stresses.Physiological and Molecular Plant Pathology,74(5/6):394-402.
Yang S D,Seo P J,Yoon H K,et al.2011.The Arabidopsis NAC transcription factor VNI2 integrates abscisic acid signals into leaf senescence via the COR/RD genes.The Plant Cell,23(6):2155-2168.
Zhong R,Lee C,Ye Z H.2010.Global analysis of direct targets of secondary wall NAC master switches in Arabidopsis.Molecular Plant,3(6):1087-1103.
王玉成,薄海侠,杨传平.2003.胡杨、柽柳总RNA提取方法的建立.东北林业大学学报,(5):99-100.(Wang Y C,Bo H Y,Yang C P.2003.A method for rapid isolation of total RNA from Tamarix and Populus euphratica Oliv.Journal of Northeast Forestry University,(5):99-100.[in Chinese])
杨勇,田新权,刘会利,等.2017.陆地棉NAC转录因子基因GhNAC6的克隆、表达和耐盐性分析.棉花学报,29(2):138-146.(Yang Y,Tian X Q,Liu H L,et al.2017.Cloning,expression and salt-tolerance analysis of the NAC transcription factor gene GhNAC6 in Upland Cotton.Cotton Science,29(2):138-146.[in Chinese])
尹林克.1995.中亚荒漠生态系统中的关键种—柽柳(Tamarix spp).干旱区研究,12(3):43-47.(Yin L K.1995.Tamarix spp.—The key species of desert ecosystem.Arid Zone Research,12(3):43-47.[in Chinese])
袁义杭,张鹤华,游韩莉,等.2018.青杄PwNAC42基因的克隆及表达模式分析.生物技术通报,34(4):1-6.(Yuan Y H,Zhang H H,You H L,et al.2018.Cloning and expression analysis of PwNAC42 in Picea wilsonii.Biotechnology Bulletin,34(4):1-6.[in Chinese])
Chen Q,Niu F,Yan J,et al.2017.Oilseed rape NAC56 transcription factor modulates reactive oxygen species accumulation and hypersensitive response-like cell death.Physiol Plant,160(2):209-221.
Duval M,Hsieh T F,Kim S Y,et al.2002.Molecular characterization of AtNAMa:member of the Arabidopsis NAC domain superfamily.Plant Molecular Biology,50(2):237-248.
Fang Y,Liao K,Du H,et al.2015.A stress-responsive NAC transcription factor SNAC3 confers heat and drought tolerance through modulation of reactive oxygen species in rice.J Exp Bot,66(21):6803-6817.
Hao Y J,Wei W,Song Q X,et al.2011.Soybean NAC transcription factors promote abiotic stress tolerance and lateral root formation in transgenic plants.The Plant Journal,68(2):302-313.
Hu H H,Dai M Q,Yao J L,et al.2006.Overexpressing a NAM,ATAF,and CUC (NAC) transcription factor enhances drought resistance and salt tolerance in rice.Proc Natl Acad Sci USA,103(35):12987-12992.
Huang D,Wang S,Zhang B,et al.2015.A gibberellin-mediated DELLA-NAC signaling cascade regulates cellulose synthesis in rice.Plant Cell,27(6):1681-1696.
Ji X Y,Zheng L,Liu Y J,et al.2014.A transient transformation system for the functional characterization of genes involved in stress response.Plant Mol Biol Rep,32(3):732-739.
Kjaersgaard T,Jensen M K,Christiansen M W,et al.2011.Senescence-associated barley NAC (NAM,ATAF1,2,CUC) transcription factor interacts with radical-induced cell death 1 through a disordered regulatory domain.Journal of Biological Chemistry,286(41):35418-35429.
Livak K J,Schmittgen T D.2001.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method.Methods,25(4):402-408.
Mao X,Zhang H,Qian X,et al.2012.TaNAC2,a NAC-type wheat transcription factor conferring enhanced multiple abiotic stress tolerances in Arabidopsis.Journal of Experimental Botany,63(8):2933-2946.
Nakashima K,Takasaki H,Mizoi J,et al.2012.NAC transcription factors in plant abiotic stress responses.Biochimica et Biophysica Acta-Gene Regulatory Mechanisma,1819(2):97-103.
Niu F,Wang C,Yan J,et al.2016.Functional characterization of NAC55 transcription factor from oilseed rape (Brassica napus L.) as a novel transcription alactivator modulating reactive oxygen species accumulation and cell death.Plant Mol Biol,92(1/2):89-104.
Ooka H,Satoh K,Doi K,et al.2003.Comprehensive analysis of NAC family genes in Oryza sativa and Arabidopsis thaliana.DNA Research,10(6):239-247.
Shen H,Yin Y,Chen F,et al.2009.A bioinformatic analysis of NAC genes for plant cell wall development in relation to lignocellulosic bioenergy production.Bioenergy Research,2(4):217-232.
Tang Y M,Liu M Y,Gao S Q,et al.2012.Molecular characterization of novel TaNAC genes in wheat and overexpression of TaNAC2a confers drought tolerance in tobacco.Physiol Plant,114(3):210-224.
Tran L S,Nakashima K,Sakuma Y,et al.2004.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 1 promoter.Plant Cell,16(9):2481-2498.
Tran L S,Nishiyama R,Yamaguchi-Shinozaki K,et al.2010.Potential utilization of NAC transcription factors to enhance abiotic stress tolerance in plants biotechnological approach.GM Crops,1(1):32-39.
Wang L,Li Z,Lu M,et al.2017b.ThNAC13,a NAC transcription factor from Tamarix hispida,confers salt and osmotic stress tolerance to transgenic Tamarix and Arabidopsis.Front Plant Sci,8:635.
Wang L L,Hu Z L,Zhu M K,et al.2017a.The abiotic stress-responsive NAC transcription factor SlNAC11 is involved in drought and salt response in tomato (Solanum lycopersicum L.).Plant Cell Tissue and Organ Culture,129(1):161-174.
Wang Z,Dane F.2013.NAC (NAM/ATAF/CUC) transcription factors in different stresses and their signaling pathway.Acta Physiologiae Plantarum,35(5):1397-1408.
Xia N,Zhang G,Liu X Y,et al.2010a.Characterization of a novel wheat NAC transcription factor gene involved in defense response against stripe rust pathogen infection and abiotic stresses.Molecular Biology Reports,37(8):3703-3712.
Xia N,Zhang G,Sun Y F,et al.2010b.TaNAC8,a novel NAC transcription factor gene in wheat,responds to stripe rust pathogen infection and abiotic stresses.Physiological and Molecular Plant Pathology,74(5/6):394-402.
Yang S D,Seo P J,Yoon H K,et al.2011.The Arabidopsis NAC transcription factor VNI2 integrates abscisic acid signals into leaf senescence via the COR/RD genes.The Plant Cell,23(6):2155-2168.
Zhong R,Lee C,Ye Z H.2010.Global analysis of direct targets of secondary wall NAC master switches in Arabidopsis.Molecular Plant,3(6):1087-1103.