烟草β-微管蛋白基因NtTubB的克隆及逆境响应分析
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摘要
为明确烟草NtTubB在烟草中的生物学功能,在烟草K326中同源克隆到1个TubB蛋白基因NtTubB,并对其基因结构、生物信息学预测、进化关系以及逆境下的表达模式进行分析。结果表明,NtTubB基因cDNA全长为2 783 bp,其中开放阅读框2 475 bp,由3个外显子和2个内含子组成,编码443个氨基酸残基组成的亲水蛋白。NtTubB蛋白序列无信号肽和跨膜结构域,存在多个磷酸化位点,主要定位于细胞质中,序列高度保守,尤其是N端序列,含有2个保守结构域(GTPase domain和C-terminal domain),均属于Tubulin/FtsZ family家族。高级结构以α-螺旋和无规卷曲为主。进化分析表明,普通烟草NtTubB的氨基酸序列与林烟草NsTubB高度同源。逆境响应分析表明,NtTubB基因受脱落酸(ABA)、水杨酸(SA)、赤霉素(GA)、茉莉酸甲酯(MeJA)、过氧化氢(H2O2)、高盐、低温和高温诱导,暗示烟草NtTubB基因参与了多种激素和非生物胁迫的应答反应。
To identify the biological function of NtTubB in tobacco, TubB protein gene NtTubB was homologously cloned in tobacco K326. The gene structure, bioinformatics prediction, evolutionary relationship and expression pattern under abiotic stress of NtTubB were analyzed. The results showed that the cDNA sequence of NtTubB was2 783 bp in full length with an open reading frame(ORF) of 2 475 bp(contained three exons and two introns), encoding a hydrophilic protein consisting of 443 amino acids residues. NtTubB protein sequence had no signal peptide and transmembrane domain, and contained multiple phosphorylation sites, mainly located in cytoplasm. The protein sequence was highly conserved, especially the N-terminal sequence, and it contained two conserved domains(GTPase domain and C-terminal domain), which belonged to the Tubulin/FtsZ family. In addition, the advanced structure was mainly based on alpha helix and random coil. Evolutionary analysis showed that the amino acid sequence of NtTubB in common tobacco and Ns TubB in Nicotiana sylvestris were highly similar. Abiotic stress response analysis showed that NtTubB gene was induced by ABA, SA, GA, MeJA, H2 O2, high salinity,low temperature and high temperature, which indicated that NtTubB might be involved in response to various hormones and abiotic stresses.
To identify the biological function of NtTubB in tobacco, TubB protein gene NtTubB was homologously cloned in tobacco K326. The gene structure, bioinformatics prediction, evolutionary relationship and expression pattern under abiotic stress of NtTubB were analyzed. The results showed that the cDNA sequence of NtTubB was2 783 bp in full length with an open reading frame(ORF) of 2 475 bp(contained three exons and two introns), encoding a hydrophilic protein consisting of 443 amino acids residues. NtTubB protein sequence had no signal peptide and transmembrane domain, and contained multiple phosphorylation sites, mainly located in cytoplasm. The protein sequence was highly conserved, especially the N-terminal sequence, and it contained two conserved domains(GTPase domain and C-terminal domain), which belonged to the Tubulin/FtsZ family. In addition, the advanced structure was mainly based on alpha helix and random coil. Evolutionary analysis showed that the amino acid sequence of NtTubB in common tobacco and Ns TubB in Nicotiana sylvestris were highly similar. Abiotic stress response analysis showed that NtTubB gene was induced by ABA, SA, GA, MeJA, H2 O2, high salinity,low temperature and high temperature, which indicated that NtTubB might be involved in response to various hormones and abiotic stresses.
引文
Abdrakhamanova A.,Wang Q.Y.,Khokhlova L.,and Nick P.,2003,Is microtubule disassembly a trigger for cold acclimation,Plant Cell Physiol.,44(7):676-686
Abe M.,Shibaoka H.,Yamane H.,and Takahashi N.,1990,Cell cycle-dependent disruption of microtubules by methyl jasmonate in tobacco BY-2 cells,Protoplasma,156(1-2):1-8
BinarováP.,CenklováV.,ProcházkováJ.,DoskocilováA.,Volc J.,Vrlík M.,and Bogre L.,2006,Gamma-tubulin is essential for acentrosomal microtubule nucleation and coordination of late mitotic events in Arabidopsis,Plant Cell,18(5):1199-1212
Breviario D.,and Nick P.,2000,Plant tubulins:a melting pot for basic questions and promising applications,Transgenic Res.,9(6):383-393
Chen Q.,Zhuo W.,Li J.H.,Lu L.M.,and Li L.Q.,2018,Cloning,bioinformatics and expression pattern analysis of Nt14-3-3,Yancao Keji(Tobacco Science&Technology),51(1):1-7(陈倩,卓维,李佳皓,鲁黎明,李立芹,2018,烟草Nt14-3-3基因的克隆及生物信息学和表达模式分析,烟草科技,51(1):1-7)
Dutcher S.K.,2001,The tubulin fraternity:alpha to eta,Curr.Opin.Cell Biol.,13(1):49-54
Fukuda M.,Hasezawa S.,Asai N.,Nakajima N.,and Kondo N.,1998,Dynamic organization of microtubules in guard cells of Vicia faba L.with diurnal cycle,Plant Cell Physiol.,39(1):80-86
Gehring C.A.,Irving H.R.,Mcconchie R.,and Parish R.W.,1997,Jasmonates induce intracellular alkalinization and closure of paphiopedilum guard cells,Ann.Bot.,80(4):485-489
Hamada T.,2007,Microtubule-associated proteins in higher plants,J.Plant Res.,120(1):79-98
Hussey P.J.,and Hawkins T.J.,2001,Plant microtubule-associated proteins:the HEAT is off in temperature-sensitive mor1,Trends Plant Sci.,6(9):389-392
Hussey P.J.,Haas N.A.,Hunsperger J.,Larkin J.,Snustad D.P.,and Silflow C.D.,1990,The beta-tubulin gene family in Zea mays:two differentially expressed beta-tubulin genes,Plant Mol.Biol.,15(6):957-972
Janke C.,and Bulinski J.C.,2011,Post-translational regulation of the microtubule cytoskeleton:mechanisms and functions,Nat.Rev.Mol.Cell Biol.,12(12):773-786
Jian L.C.,Sun L.H.,and Lin Z.P.,1989,Studies on microtubule cold stability in relation to plant cold hardiness,Zhiwu Xuebao(Acta Botanica Sinica),31(10):737-741(简令成,孙龙华,林忠平,1989,微管的冷稳定性与植物抗寒性关系的研究,植物学报,31(10):737-741)
Kopczak S.D.,Haas N.A.,Hussey P.J.,Silflow C.D.,and Snustad D.P.,1992,The small genome of Arabidopsis contains at least six expressed alpha-tubulin genes,Plant Cell,4(5):539-547
Ledbetter M.C.,and Porter K.R.,1963,A"microtubule"in plant cell fine structure,J.Cell Biol.,19:239-250
Li Q.Y.,Wang S.S.,Zhou P.L.,Liu G.L.,Zeng W.L.,Zhou D.J.,Zheng X.and Chen A.G.,2018,Effects of low temperature stress on leaf shape and physiological characteristics in tobacco seedlings,Zhongguo Yancao Kexue(Chinese Tobacco Science),39(1):17-23(李琦瑶,王树声,周培禄,刘光亮,曾文龙,周道金,郑璇,陈爱国,2018,低温胁迫对烟苗叶形及生理特性的影响,中国烟草科学,39(1):17-23)
Li R.Y.,Islam S.U.,Wu Z.J.,and Ye X.J.,2016,Bensulfuron-methyl treatment of soil sffects the infestation of whitefly,aphid,and toba cco mosaic viruson Nicotiana tabacum,Front.Plant Sci.,7(2):1954-1970
Livak K.J.,and Schmittgen T.D.,2001,Analysis of relative gene expression data using real-time quantitative PCR and the2-ΔΔCTmethod,Methods,25(4):402-408
Luo X.P.,Zhu D.Y.,Huang Y.J.,Li M.F.,Yao P.F.,Gao F.,Li C.L.,and Zhao H.X.,2015,Effects of methyl jasmonate accumulation of flavonoids and related gene expression of buckwheat sprouts,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(5):1040-1046(雒晓鹏,朱冬寅,黄云吉,李茂菲,姚攀锋,高飞,李成磊,赵海霞,2015,茉莉酸甲酯对芽期苦荞黄酮积累及相关基因表达的影响,基因组学与应用生物学,34(5):1040-1046)
Luo X.Y.,2015,Studies on cloning and expression characterization of Ntpx in tobacco,Thesis for M.S.,Hunan Agricultural University,Supervisor:Tian Y.,and Lu X.Y.,pp.23-24(罗秀云,2015,烟草中Ntpx基因的克隆及表达特性研究,硕士学位论文,湖南农业大学,导师:田云,卢向阳,pp.23-24)
Mazars C.,Thion L.,Thuleau P.,Graziana A.,Knight M.R.,Moreau M.,and Ranjeva R.,1997,Organization of cytoskeleton controls the changes in cytosolic calcium of coldshocked Nicotiana plumbaginifolia protoplasts,Cell Calcium,22(5):413-420
Mckean P.G.,Vaughan S.,and Gull K.,2001,The extended tubulin superfamily,J.Cell Sci.,114(15):2723-2733
Oakley B.R.,2000,An abundance of tubulins,Trends in Cell Biology,10(12):537-542
Pastuglia M.,Azimzadeh J.,Goussot M.,Camilleri C.,Belcram K.,Evrard J.L.,Schmit A.C.,Guerche P.,and Bouchez D.,2006,Gamma-tubulin is essential for microtubule organization and development in Arabidopsis,Plant Cell,18(6):1412-1425
Rao G.D.,and Zhang J.G.,2013,Advances of studies on plant tubulin gene,Shijie Linye Yanjiu(World Forestry Research),26(3):17-20(饶国栋,张建国,2013,植物微管蛋白基因研究进展,世界林业研究,26(3):17-20)
Rikin A.,Dan A.,and Gitler C.,1980,Chilling injury in cotton(Gossypium hirsutum L.):Effects of antimicrotubular drugs,Plant Cell Physiol.,21(5):829-837
Sangwan V.,Foulds I.,Singh J.,and Dhindsa R.S.,2001,Cold-activation of Brassica napus BN115 promoter is mediated by structural changes in membranes and cytoskeleton,and requires Ca2+influx,Plant J.,27(1):1-12
Schmidt G.W.,and Delaney S.K.,2010,Stable internal reference genes for normalization of real-time RT-PCR in tobacco(Nicotiana tabacum)during development and abiotic stress,Mol.Genet Genomics,283(3):233-241
Smallwood M.,and Bowles D.J.,2002,Plants in a cold climate,Phil.Trans.R.Soc.Lond.B.,357(1423):831-847
Snustad D.P.,Haas N.A.,Kopczak S.D.,and Silflow C.D.,1992,The small genome of Arabidopsis contains at least nine expressed beta-tubulin genes,Plant Cell,4(5):549-556
Villemur R.,Haas N.A.,Joyce C.M.,Snustad D.P.,and Silflow C.D.,1994,Characterization of four new beta-tubulin genes and their expression during male flower development in maize(Zea mays L.),Plant Mol.Biol.,24(2):295-315
Villemur R.,Joyce C.M.,Haas N.A.,Goddard R.H.,Kopczak S.D.,Hussey P.J.,Snustad D.P.,and Silflow C.D.,1992,Alpha-tubulin gene family of maize(Zea mays L.).Evidence for two ancient alpha-tubulin genes in plants,J.Mol.Biol.,227(1):81-96
Wang C.,Li J.J.,and Yuan M.,2007,Salt tolerance requires cortical microtubule reorganization in Arabidopsis,Plant Cell Physiol.,48(11):1534-1547
Wang Q.,Yang K.,and Li Y.H.,2005,Microtubule organizing centers and related proteins in higher plants,Zhiwu Shenglixue Tongxun(Plant Physiology Communications),41(3):400-404(王琦,杨坤,李艳红,2005,高等植物微管组织中心及其相关蛋白,植物生理学通讯,41(3):400-404)
Wang Q.,Zhao S.,Wang L.,Wang X.D.,Yang K.,Wen G.L.,Zhuang M.,Wang X.W.,and Li Y.H.,2007,Tobaccoγ-tubulin gene silencing mediated by recombinant potato virus X vector,Zhiwu Shengli Yu Fenzi Shengwuxue Xuebao(Journal of Plant Physiology and Molecular Biology),33(5):375-386(王琦,赵爽,王璐,王晓丹,杨坤,温国蕾,庄木,王晓武,李艳红,2007,重组马铃薯X病毒载体介导的烟草γ-微管蛋白基因沉默,植物生理与分子生物学学报,33(5):375-386)
Xu L.,Huang L.P.,Lu L.M.,and Li L.Q.,2017,Cloning and expression analysis of potassium channel NtTPK gene in tobacco,Zhejiang Nongye Xuebao(Acta Agriculturae Zhejiangen-sis),29(3):366-372(许力,黄路平,鲁黎明,李立芹,2017,烟草钾离子通道基因NtTPK的克隆及表达分析,浙江农业学报,29(3):366-372)
Yang H.,Deng Z.,Liu H.,Dai L.J.,and Li D.J.,2017,Cloning,expression and bioinformatics analysis of HbTUA2 gene in Hevea brasiliensis(rubber tree),Zhiwu Shengli Xuebao(Plant Physiology Journal),53(1):52-62(杨洪,邓治,刘辉,代龙军,李德军,2017,巴西橡胶树α-微管蛋白HbTUA2基因的克隆,表达及生物信息学分析,植物生理学报,53(1):52-62)
Zhang Q.,Lin F.,Mao T.,Nie J.,Yan M.,Yuan M.,and Zhang W.,2012,Phosphatidic acid regulates microtubule organization by interacting with MAP65-1 in response to salt stress in Arabidopsis,Plant Cell,24(11):4555-4576
Zhao S.,Yang K.,Ma Q.,Wang Q.,Wang X.D.,and Li Y.H.,2009,Plant polar growth in tobacco disturbed byγ-tubulin gene silencing,Prog.Nat.Sci-Mater.,19(6):685-691
Zhu J.K.,2002,Salt and drought stress signal transduction in plants,Annu.Rev.Plant Biol.,53(53):247-273
Abe M.,Shibaoka H.,Yamane H.,and Takahashi N.,1990,Cell cycle-dependent disruption of microtubules by methyl jasmonate in tobacco BY-2 cells,Protoplasma,156(1-2):1-8
BinarováP.,CenklováV.,ProcházkováJ.,DoskocilováA.,Volc J.,Vrlík M.,and Bogre L.,2006,Gamma-tubulin is essential for acentrosomal microtubule nucleation and coordination of late mitotic events in Arabidopsis,Plant Cell,18(5):1199-1212
Breviario D.,and Nick P.,2000,Plant tubulins:a melting pot for basic questions and promising applications,Transgenic Res.,9(6):383-393
Chen Q.,Zhuo W.,Li J.H.,Lu L.M.,and Li L.Q.,2018,Cloning,bioinformatics and expression pattern analysis of Nt14-3-3,Yancao Keji(Tobacco Science&Technology),51(1):1-7(陈倩,卓维,李佳皓,鲁黎明,李立芹,2018,烟草Nt14-3-3基因的克隆及生物信息学和表达模式分析,烟草科技,51(1):1-7)
Dutcher S.K.,2001,The tubulin fraternity:alpha to eta,Curr.Opin.Cell Biol.,13(1):49-54
Fukuda M.,Hasezawa S.,Asai N.,Nakajima N.,and Kondo N.,1998,Dynamic organization of microtubules in guard cells of Vicia faba L.with diurnal cycle,Plant Cell Physiol.,39(1):80-86
Gehring C.A.,Irving H.R.,Mcconchie R.,and Parish R.W.,1997,Jasmonates induce intracellular alkalinization and closure of paphiopedilum guard cells,Ann.Bot.,80(4):485-489
Hamada T.,2007,Microtubule-associated proteins in higher plants,J.Plant Res.,120(1):79-98
Hussey P.J.,and Hawkins T.J.,2001,Plant microtubule-associated proteins:the HEAT is off in temperature-sensitive mor1,Trends Plant Sci.,6(9):389-392
Hussey P.J.,Haas N.A.,Hunsperger J.,Larkin J.,Snustad D.P.,and Silflow C.D.,1990,The beta-tubulin gene family in Zea mays:two differentially expressed beta-tubulin genes,Plant Mol.Biol.,15(6):957-972
Janke C.,and Bulinski J.C.,2011,Post-translational regulation of the microtubule cytoskeleton:mechanisms and functions,Nat.Rev.Mol.Cell Biol.,12(12):773-786
Jian L.C.,Sun L.H.,and Lin Z.P.,1989,Studies on microtubule cold stability in relation to plant cold hardiness,Zhiwu Xuebao(Acta Botanica Sinica),31(10):737-741(简令成,孙龙华,林忠平,1989,微管的冷稳定性与植物抗寒性关系的研究,植物学报,31(10):737-741)
Kopczak S.D.,Haas N.A.,Hussey P.J.,Silflow C.D.,and Snustad D.P.,1992,The small genome of Arabidopsis contains at least six expressed alpha-tubulin genes,Plant Cell,4(5):539-547
Ledbetter M.C.,and Porter K.R.,1963,A"microtubule"in plant cell fine structure,J.Cell Biol.,19:239-250
Li Q.Y.,Wang S.S.,Zhou P.L.,Liu G.L.,Zeng W.L.,Zhou D.J.,Zheng X.and Chen A.G.,2018,Effects of low temperature stress on leaf shape and physiological characteristics in tobacco seedlings,Zhongguo Yancao Kexue(Chinese Tobacco Science),39(1):17-23(李琦瑶,王树声,周培禄,刘光亮,曾文龙,周道金,郑璇,陈爱国,2018,低温胁迫对烟苗叶形及生理特性的影响,中国烟草科学,39(1):17-23)
Li R.Y.,Islam S.U.,Wu Z.J.,and Ye X.J.,2016,Bensulfuron-methyl treatment of soil sffects the infestation of whitefly,aphid,and toba cco mosaic viruson Nicotiana tabacum,Front.Plant Sci.,7(2):1954-1970
Livak K.J.,and Schmittgen T.D.,2001,Analysis of relative gene expression data using real-time quantitative PCR and the2-ΔΔCTmethod,Methods,25(4):402-408
Luo X.P.,Zhu D.Y.,Huang Y.J.,Li M.F.,Yao P.F.,Gao F.,Li C.L.,and Zhao H.X.,2015,Effects of methyl jasmonate accumulation of flavonoids and related gene expression of buckwheat sprouts,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(5):1040-1046(雒晓鹏,朱冬寅,黄云吉,李茂菲,姚攀锋,高飞,李成磊,赵海霞,2015,茉莉酸甲酯对芽期苦荞黄酮积累及相关基因表达的影响,基因组学与应用生物学,34(5):1040-1046)
Luo X.Y.,2015,Studies on cloning and expression characterization of Ntpx in tobacco,Thesis for M.S.,Hunan Agricultural University,Supervisor:Tian Y.,and Lu X.Y.,pp.23-24(罗秀云,2015,烟草中Ntpx基因的克隆及表达特性研究,硕士学位论文,湖南农业大学,导师:田云,卢向阳,pp.23-24)
Mazars C.,Thion L.,Thuleau P.,Graziana A.,Knight M.R.,Moreau M.,and Ranjeva R.,1997,Organization of cytoskeleton controls the changes in cytosolic calcium of coldshocked Nicotiana plumbaginifolia protoplasts,Cell Calcium,22(5):413-420
Mckean P.G.,Vaughan S.,and Gull K.,2001,The extended tubulin superfamily,J.Cell Sci.,114(15):2723-2733
Oakley B.R.,2000,An abundance of tubulins,Trends in Cell Biology,10(12):537-542
Pastuglia M.,Azimzadeh J.,Goussot M.,Camilleri C.,Belcram K.,Evrard J.L.,Schmit A.C.,Guerche P.,and Bouchez D.,2006,Gamma-tubulin is essential for microtubule organization and development in Arabidopsis,Plant Cell,18(6):1412-1425
Rao G.D.,and Zhang J.G.,2013,Advances of studies on plant tubulin gene,Shijie Linye Yanjiu(World Forestry Research),26(3):17-20(饶国栋,张建国,2013,植物微管蛋白基因研究进展,世界林业研究,26(3):17-20)
Rikin A.,Dan A.,and Gitler C.,1980,Chilling injury in cotton(Gossypium hirsutum L.):Effects of antimicrotubular drugs,Plant Cell Physiol.,21(5):829-837
Sangwan V.,Foulds I.,Singh J.,and Dhindsa R.S.,2001,Cold-activation of Brassica napus BN115 promoter is mediated by structural changes in membranes and cytoskeleton,and requires Ca2+influx,Plant J.,27(1):1-12
Schmidt G.W.,and Delaney S.K.,2010,Stable internal reference genes for normalization of real-time RT-PCR in tobacco(Nicotiana tabacum)during development and abiotic stress,Mol.Genet Genomics,283(3):233-241
Smallwood M.,and Bowles D.J.,2002,Plants in a cold climate,Phil.Trans.R.Soc.Lond.B.,357(1423):831-847
Snustad D.P.,Haas N.A.,Kopczak S.D.,and Silflow C.D.,1992,The small genome of Arabidopsis contains at least nine expressed beta-tubulin genes,Plant Cell,4(5):549-556
Villemur R.,Haas N.A.,Joyce C.M.,Snustad D.P.,and Silflow C.D.,1994,Characterization of four new beta-tubulin genes and their expression during male flower development in maize(Zea mays L.),Plant Mol.Biol.,24(2):295-315
Villemur R.,Joyce C.M.,Haas N.A.,Goddard R.H.,Kopczak S.D.,Hussey P.J.,Snustad D.P.,and Silflow C.D.,1992,Alpha-tubulin gene family of maize(Zea mays L.).Evidence for two ancient alpha-tubulin genes in plants,J.Mol.Biol.,227(1):81-96
Wang C.,Li J.J.,and Yuan M.,2007,Salt tolerance requires cortical microtubule reorganization in Arabidopsis,Plant Cell Physiol.,48(11):1534-1547
Wang Q.,Yang K.,and Li Y.H.,2005,Microtubule organizing centers and related proteins in higher plants,Zhiwu Shenglixue Tongxun(Plant Physiology Communications),41(3):400-404(王琦,杨坤,李艳红,2005,高等植物微管组织中心及其相关蛋白,植物生理学通讯,41(3):400-404)
Wang Q.,Zhao S.,Wang L.,Wang X.D.,Yang K.,Wen G.L.,Zhuang M.,Wang X.W.,and Li Y.H.,2007,Tobaccoγ-tubulin gene silencing mediated by recombinant potato virus X vector,Zhiwu Shengli Yu Fenzi Shengwuxue Xuebao(Journal of Plant Physiology and Molecular Biology),33(5):375-386(王琦,赵爽,王璐,王晓丹,杨坤,温国蕾,庄木,王晓武,李艳红,2007,重组马铃薯X病毒载体介导的烟草γ-微管蛋白基因沉默,植物生理与分子生物学学报,33(5):375-386)
Xu L.,Huang L.P.,Lu L.M.,and Li L.Q.,2017,Cloning and expression analysis of potassium channel NtTPK gene in tobacco,Zhejiang Nongye Xuebao(Acta Agriculturae Zhejiangen-sis),29(3):366-372(许力,黄路平,鲁黎明,李立芹,2017,烟草钾离子通道基因NtTPK的克隆及表达分析,浙江农业学报,29(3):366-372)
Yang H.,Deng Z.,Liu H.,Dai L.J.,and Li D.J.,2017,Cloning,expression and bioinformatics analysis of HbTUA2 gene in Hevea brasiliensis(rubber tree),Zhiwu Shengli Xuebao(Plant Physiology Journal),53(1):52-62(杨洪,邓治,刘辉,代龙军,李德军,2017,巴西橡胶树α-微管蛋白HbTUA2基因的克隆,表达及生物信息学分析,植物生理学报,53(1):52-62)
Zhang Q.,Lin F.,Mao T.,Nie J.,Yan M.,Yuan M.,and Zhang W.,2012,Phosphatidic acid regulates microtubule organization by interacting with MAP65-1 in response to salt stress in Arabidopsis,Plant Cell,24(11):4555-4576
Zhao S.,Yang K.,Ma Q.,Wang Q.,Wang X.D.,and Li Y.H.,2009,Plant polar growth in tobacco disturbed byγ-tubulin gene silencing,Prog.Nat.Sci-Mater.,19(6):685-691
Zhu J.K.,2002,Salt and drought stress signal transduction in plants,Annu.Rev.Plant Biol.,53(53):247-273