烟草谷氧还蛋白NbGRX1的亚细胞定位及抗旱功能研究
|
摘要
[目的]本文旨在分析烟草NbGRX1基因的亚细胞定位及抗旱功能。[方法]在前期研究中,我们从烟草中克隆了1个谷氧还蛋白基因NbGRX1,并对其表达特性进行了研究。在此基础上,对NbGRX1蛋白的亚细胞定位及抗旱功能进行研究。利用病毒介导的基因沉默,在烟草中沉默了该基因,并调查NbGRX1基因沉默后烟草对干旱的响应。同时采用农杆菌介导法,将NbGRX1基因转化至拟南芥中,筛选单拷贝插入的纯合转基因拟南芥进行后续研究,对转基因拟南芥及对照进行干旱处理,并分析其表型。[结果]亚细胞定位研究表明,空载体中的GFP(绿色荧光蛋白)定位在细胞质和细胞核中,GFP:NbGRX1的融合蛋白均匀分布在细胞质和细胞核中,因此可以推测NbGRX1蛋白能定位在植物的细胞核和细胞质中。病毒介导的基因沉默研究表明,NbGRX1基因沉默烟草与对照相比对干旱胁迫更为敏感,更容易出现黄化和萎蔫的症状,叶片相对含水量下降;NbGRX1基因超量表达的株系干旱能力明显高于对照。[结论]烟草NbGRX1基因能够提高植物的抗旱性。
[Objectives]The aim of this paper is to understand the subcellular localization of NbGRX1( Nicotiana benthamiana glutaredoxin 1) and its role in plant response to drought stress. [Methods]To investigate the subcellular localization of NbGRX1 protein in plant cell,the NbGRX1 protein was fused to the C-terminus of GFP( green fluorescent protein) and the construct was transiently expressed in N. benthamiana leaf epidermal cells and then monitored by capturing GFP fluorescence. To uncover the function of the NbGRX1 gene in response to drough stress,NbGRX1 gene silencing vector was constructed based on the geminivirus satellite silencing vector and then introduced into Agrobacterium tumefaciens strain EHA105. NbGRX1-silenced and non-silenced control plants were imposed by withholding water in a growth chamber until wilted leaves were observed on all the NbGRX1-silenced plants and relative water content( RWC) in the leaves of each plant was also estimated. Next,the coding sequence of NbGRX1 was introduced into Arabidopsis under the control of the cauliflower mosaic virus( Ca MV) 35 S promoter. Independent homozygous lines with a single copy of NbGRX1 were allowed to grow for 3 generations for further studies. F_or drought assay,plants were imposed by withholding water in a growth chamber until a lethal effect of dehydration was observed on all of the control plants. Plants treated with water were regarded as the mock. [Results]The results suggested that NbGRX1 protein was localized in both nucleus and cytoplasm. Virus-induced gene silencing of NbGRX1 in tobacco led to increased sensitivity to drought stress with easily becoming yellowing and wilt with decreased relative water content,suggesting that NbGRX1 was required for plant resistance to drought stress. The coding sequence of NbGRX1 was introduced into Arabidopsis and two independent homozygous lines( N1,N2) with a single copy of NbGRX1 were allowed to grow for 3 generations for further studies. Over-expression of NbGRX1 in Arabidopsis plants enhanced the lines in their tolerance to drought in soil-grown conditions,conforming that NbGRX1 plays important role in plant resistance to drought stress. [Conclusions]Our research demonstrated that NbGRX1 can improve plant resistance to drought stress.
[Objectives]The aim of this paper is to understand the subcellular localization of NbGRX1( Nicotiana benthamiana glutaredoxin 1) and its role in plant response to drought stress. [Methods]To investigate the subcellular localization of NbGRX1 protein in plant cell,the NbGRX1 protein was fused to the C-terminus of GFP( green fluorescent protein) and the construct was transiently expressed in N. benthamiana leaf epidermal cells and then monitored by capturing GFP fluorescence. To uncover the function of the NbGRX1 gene in response to drough stress,NbGRX1 gene silencing vector was constructed based on the geminivirus satellite silencing vector and then introduced into Agrobacterium tumefaciens strain EHA105. NbGRX1-silenced and non-silenced control plants were imposed by withholding water in a growth chamber until wilted leaves were observed on all the NbGRX1-silenced plants and relative water content( RWC) in the leaves of each plant was also estimated. Next,the coding sequence of NbGRX1 was introduced into Arabidopsis under the control of the cauliflower mosaic virus( Ca MV) 35 S promoter. Independent homozygous lines with a single copy of NbGRX1 were allowed to grow for 3 generations for further studies. F_or drought assay,plants were imposed by withholding water in a growth chamber until a lethal effect of dehydration was observed on all of the control plants. Plants treated with water were regarded as the mock. [Results]The results suggested that NbGRX1 protein was localized in both nucleus and cytoplasm. Virus-induced gene silencing of NbGRX1 in tobacco led to increased sensitivity to drought stress with easily becoming yellowing and wilt with decreased relative water content,suggesting that NbGRX1 was required for plant resistance to drought stress. The coding sequence of NbGRX1 was introduced into Arabidopsis and two independent homozygous lines( N1,N2) with a single copy of NbGRX1 were allowed to grow for 3 generations for further studies. Over-expression of NbGRX1 in Arabidopsis plants enhanced the lines in their tolerance to drought in soil-grown conditions,conforming that NbGRX1 plays important role in plant resistance to drought stress. [Conclusions]Our research demonstrated that NbGRX1 can improve plant resistance to drought stress.
引文
[1]VranováE,InzéD,van Breusegem F.Signal transduction during oxidative stress[J].Journal of Experimental Botany,2002,53:1227-1236.
[2]Buchanan B B,Balmer Y.Redox regulation:a broadening horizon[J].Annual Review of Plant Biology,2005,56:187-220.
[3]Scandalios J G.The rise of ROS[J].Trends in Biochemical Science,2002,27:483-486.
[4]Pitzschke A,Forzani C,Hirt H.Reactive oxygen species signaling in plants[J].Antioxidants and Redox Signaling,2006,8:1757-1764.
[5]Rouhier N.Plant glutaredoxins:pivotal players in redox biology and iron-sulphur centre assembly centre assembly[J].New Phytologist,2010,186:365-372.
[6]Dutilleul C,Garmier M,Noctor G,et al.Leaf mitochondria modulate whole cell redox homeostasis,set antioxidant capacity,and determine stress resistance through altered signaling and diurnal regulation[J].Plant Cell,2003,15:1212-1226.
[7]Foyer C H,Noctor G.Redox homeostasis and antioxidant signaling:a metabolic interface between stress perception and physiological responses[J].Plant Cell,2005,17:1866-1875.
[8]郭玉双,张建华,张吉顺,等.烟草谷氧还蛋白基因Nb GRX1的克隆与表达特性分析[J].东北农业大学学报,2011,42(10):47-51.Guo Y S,Zhang J H,Zhang J S,et al.Cloning and expression pattern analysis a novel Nb GRX1 gene from Nicotiana benthamiana[J].Journal of Northeast Agricultural University,2011,42(10):47-51(in Chinese with English abstract).
[9]Xiong R Y,Wu J X,Zhou Y J,et al.Characterization and subcellular localization of an RNA silencing suppressor encoded by rice stripe tenuivirus[J].Virology,2009,387:29-40.
[10]Huang C J,Xie Y,Zhou X P,et al.Efficient virus-induced gene silencing in plants using a modified geminivirus DNA1 component[J].Plant Biotechnology Journal,2009,7:254-265.
[11]Turner N C.Techniques and experimental approaches for the measurement of plant water status[J].Plant and Soil,1981,58:339-366.
[12]郭玉双,陈静,刘筱嘉,等.烟草中一个LTR类反转录转座子基因的克隆与功能分析[J].南京农业大学学报,2015,38(6):936-942.DOI:10.7685/j.issn.1000-2030.2015.06.010.Guo Y S,Chen J,Liu X J,et al.Isolation and function analysis a LTR-retrotransposons from tobacco(Nicotiana tabacum)[J].Journal of Nanjing Agricultrual University,2015,38(6):936-942(in Chinese with English abstract).
[13]Cui X,Li G,Wang D,et al.A begomovirus DNA beta-encoded protein binds DNA,functions as a suppressor of RNA silencing,and targets the cell nucleus[J].Journal of Virology,2005,79:10764-10775.
[14]Xing S P,Rosso M G,Zachgo S.ROXY1,a member of the plant glutaredoxin family,is required for petal development in Arabidopsis thaliana[J].Development,2005,132:1555-1565.
[15]Li S,Lauri A,Ziemann M,et al.Nuclear activity of ROXY1,a glutaredoxin interacting with TGA factors,is required for petal development in Arabidopsis thaliana[J].Plant Cell,2009,21:429-441.
[16]Xing S,Zachgo S.ROXY1 and ROXY2,two Arabidopsis glutaredoxin genes,are required for anther development[J].Plant Journal,2008,53:790-801.
[17]Ndamukong I,Abdallat A A,Thurow C,et al.SA-inducible Arabidopsis glutaredoxin interacts with TGA factors and suppresses JA-responsive PDF1.2 transcription[J].Plant Journal,2007,50:128-139.
[18]Walters L A,Escobar M A.The At GRXS3/4/5/7/8 glutaredoxin gene cluster on Arabidopsis thaliana chromosome 4 is coordinately regulated by nitrate and appears to control primary root growth[J].Plant Signaling and Behavior,2016,11(4):e1171450.
[19]Guo Y S,Huang C J,Xie Y,et al.A tomato glutaredoxin gene Sl GRX1 regulates plant responses to oxidative,drought and salt stresses[J].Planta,2010,232:1499-1509.
[2]Buchanan B B,Balmer Y.Redox regulation:a broadening horizon[J].Annual Review of Plant Biology,2005,56:187-220.
[3]Scandalios J G.The rise of ROS[J].Trends in Biochemical Science,2002,27:483-486.
[4]Pitzschke A,Forzani C,Hirt H.Reactive oxygen species signaling in plants[J].Antioxidants and Redox Signaling,2006,8:1757-1764.
[5]Rouhier N.Plant glutaredoxins:pivotal players in redox biology and iron-sulphur centre assembly centre assembly[J].New Phytologist,2010,186:365-372.
[6]Dutilleul C,Garmier M,Noctor G,et al.Leaf mitochondria modulate whole cell redox homeostasis,set antioxidant capacity,and determine stress resistance through altered signaling and diurnal regulation[J].Plant Cell,2003,15:1212-1226.
[7]Foyer C H,Noctor G.Redox homeostasis and antioxidant signaling:a metabolic interface between stress perception and physiological responses[J].Plant Cell,2005,17:1866-1875.
[8]郭玉双,张建华,张吉顺,等.烟草谷氧还蛋白基因Nb GRX1的克隆与表达特性分析[J].东北农业大学学报,2011,42(10):47-51.Guo Y S,Zhang J H,Zhang J S,et al.Cloning and expression pattern analysis a novel Nb GRX1 gene from Nicotiana benthamiana[J].Journal of Northeast Agricultural University,2011,42(10):47-51(in Chinese with English abstract).
[9]Xiong R Y,Wu J X,Zhou Y J,et al.Characterization and subcellular localization of an RNA silencing suppressor encoded by rice stripe tenuivirus[J].Virology,2009,387:29-40.
[10]Huang C J,Xie Y,Zhou X P,et al.Efficient virus-induced gene silencing in plants using a modified geminivirus DNA1 component[J].Plant Biotechnology Journal,2009,7:254-265.
[11]Turner N C.Techniques and experimental approaches for the measurement of plant water status[J].Plant and Soil,1981,58:339-366.
[12]郭玉双,陈静,刘筱嘉,等.烟草中一个LTR类反转录转座子基因的克隆与功能分析[J].南京农业大学学报,2015,38(6):936-942.DOI:10.7685/j.issn.1000-2030.2015.06.010.Guo Y S,Chen J,Liu X J,et al.Isolation and function analysis a LTR-retrotransposons from tobacco(Nicotiana tabacum)[J].Journal of Nanjing Agricultrual University,2015,38(6):936-942(in Chinese with English abstract).
[13]Cui X,Li G,Wang D,et al.A begomovirus DNA beta-encoded protein binds DNA,functions as a suppressor of RNA silencing,and targets the cell nucleus[J].Journal of Virology,2005,79:10764-10775.
[14]Xing S P,Rosso M G,Zachgo S.ROXY1,a member of the plant glutaredoxin family,is required for petal development in Arabidopsis thaliana[J].Development,2005,132:1555-1565.
[15]Li S,Lauri A,Ziemann M,et al.Nuclear activity of ROXY1,a glutaredoxin interacting with TGA factors,is required for petal development in Arabidopsis thaliana[J].Plant Cell,2009,21:429-441.
[16]Xing S,Zachgo S.ROXY1 and ROXY2,two Arabidopsis glutaredoxin genes,are required for anther development[J].Plant Journal,2008,53:790-801.
[17]Ndamukong I,Abdallat A A,Thurow C,et al.SA-inducible Arabidopsis glutaredoxin interacts with TGA factors and suppresses JA-responsive PDF1.2 transcription[J].Plant Journal,2007,50:128-139.
[18]Walters L A,Escobar M A.The At GRXS3/4/5/7/8 glutaredoxin gene cluster on Arabidopsis thaliana chromosome 4 is coordinately regulated by nitrate and appears to control primary root growth[J].Plant Signaling and Behavior,2016,11(4):e1171450.
[19]Guo Y S,Huang C J,Xie Y,et al.A tomato glutaredoxin gene Sl GRX1 regulates plant responses to oxidative,drought and salt stresses[J].Planta,2010,232:1499-1509.