摘要
干旱、盐碱、低温和冻害等非生物逆境是影响植物生长发育的主要环境因素,严重地制约着农作物产量与品质的提高。在逆境胁迫下,植物能感知、传递胁迫信号,通过激活响应转录因子,启动抗逆功能基因的表达,使植物体内发生一系列的生理生化反应,抵御不良环境的危害,从而提高植物的耐逆性。研究表明:AP2/EREBP家族和NAC家族转录因子在抗逆应答反应中发挥着重要作用。本文从柳枝稷和普通小麦克隆抗逆相关AP2/EREBP和NAC转录因子,并对其特性和功能分析,旨在为作物的抗性育种提供科学依据。本研究取得的结果如下:
1.基因分离及序列分析:采用同源克隆方法,从柳枝稷中分离到3个AP2/EREBP和1个NAC转录因子基因全长,分别命名为PvEREBP、PvDREB1、PvDREB2、PvNAC、进化树分析显示:PvEREBP与玉米、水稻、小麦的EREBP转录因子具有较高同源性:PvDREB1、PvDREB2与玉米DREB类转录因子同源性较高;PvNAC与玉米、水稻、小麦的NAC转录因子同源性较高。从小麦中分离到1个NAC转录因子基因,命名为TaNAC,TaNAC与其它植物NAC类转录因子转录因子同源性都很低。
2.基因表达特性分析:在非生物胁迫和ABA的处理柳枝稷和小麦材料,利用荧光定量方法分析表明:PvEREBP基因不同程度的受干旱、高盐、低温和ABA诱导表达,PvDREB1仅受高盐诱导表达,PvDREB2受高盐和ABA诱导表达,PvNAC受干旱、高盐和ABA诱导表达,TaNAC受干旱、高盐、低温诱导表达。
3.亚细胞定位分析:利用含有绿色荧光蛋白的载体163-GFP构建PvEREBP和PvNAC基因的融合表达载体,采用基因枪转化法转化洋葱表皮细胞,观察到PvEREBP和PvNAC的编码蛋白有亚细胞定位功能,能定位到细胞核上。
4.转基因烟草功能分析:构建PvEREBP、PvDREB1、PvDREB2、PvNAC和TaNAC四个基因构建表达载体,利用农杆菌介导,转化烟草,并进行抗逆功能分析,结果表明:PvEREBP、PvDREB2、PvNAC和TaNAC转基因植株增强了耐盐性;PvEREBP和TaNAC转基因烟草植株提高了抗旱性。
Drought,salinity,cold and freeze are the key environment factors affecting plant growth and development,and seriously limit the increase of yield and improvement of quality of crops. Under abiotic stress,plants could sense and transduce the signals caused by stress to transcription factors through a series of phosphorization responses.A large number of studies have proved AP2/EREBP and NAC transcription factors play important roles in response to biotic and abiotic stresses.In this paper,New AP2/EREBP and NAC genes was cloned from switchgrass(Panicum virgatum L.) and wheat(Triticum aestivum),and study their characteristics and functional Identifications.
This study make the these following progresses
1.Gene Isolation and sequence analysis:Using the homology cloning methods,it was isolated three AP2/EREBP genes and a NAC gene from switchgrass which were designated PvEREBP、PvDREB1,PvDREB2、PvNAC respectively.Homology trees analysis showed that PvEREBP had higher homology with EREBP transcription factors from corn,rice and wheat,PvDREB1 and PvDREB2 had higher homology with DREB transcription factor from maize and Pennisetum glancum,PvNaC had higher homology with NAC transcription factors from corn,rice and wheat.A NAC transcription factor gene which was designated TaNAC from wheat was isolated.Homology tree analysis showed that TaNAC had low homology with other plant families transcription factors.
2.Gene expression analysis:By the method of fluorescence quantitative,PvEREBP, PvDREB1,PvDREB2,PvNAC and TaNAC genes expression was analyzed under abiotic stress and ABA.The results showed that PvEREBP gene was induced by drought,high salinity,low temperature and ABA,PvDREB1 gene only by high salt,PvDREB2 gene by high salt and ABA,PvNAC by drought,high salinity,and ABA,and TaNAC by drought, high-salt and low temperature.
3.Subcellular localization analysis:PvEREBP and PvNAC proteins were localized inside nucleus inonion epidermal cells by constructing GFP-PvEREBP and GFP-PvNAC fusion expression carriers and transformed by particle bombardment.
4.Functional analysis of transgenic tobacco:constructed four gene expression vector and transformated tobacco through the leaf discs transformation.Transgenic tobacco were treated by PEG,NaCl and ABA.The results showed that over-expression of PvERE-BP,PvDREB2, PvNAC and TaNAC improved transgenic tobacco toleranceto salt,also,over-expression of PvEREBP and TaNAC improved transgenic tobacc resistance to drought.
引文
1 Boyer JS.productivity and environment.Science,1982,218:443-448.
2 Cattivelli L,Baldi P.Chromosome regions and stress-related Sequences involved in resistance to abiotic stress in Triticeae.Plant MolecularBiology,2002,48:649-665.
3 Singh KB.Transcriptional regulation in plants:The importance of combinatorial control.Plant Physiol,1998,118:1111-1120.
4 Liu L,White M J,MacRae T H.Transcription factors and their genes in higher plants.1999,European Journal of Biochemistry,262:247-257.
5 Meshi T,Iwabuchi M.Plant transcription factors.Plant Cell Physio,1995,136(8):1405-1420.
6 Schwechheimer C,Bevan M.The regulation of transcription factor activity in plants.Trend in Plant Science,19983,(10):278-283.
7 Sainz M B,Goff S A,Chandler V L.Extensive mutagenesis of a transcriptional activation identifies single Hydrophobic and acidic amino acids important for activation in vivo.1997,Mol Cell Bin,17:115-122.
8 Boulikas T,Putative nuclear lacalization signals(NLS) in protein transcription facors.1994,Cell Biochen,55,32-58.
9 Gutierrez RA,Green PJ,Keegstra K,et al.Phylogenetic profiling of the Arabidopsis thaliana proteome:what proteins distinguish plants from other organisms?.Genome Biology,2004,5(8):R53.
10 Gu Y Q,Wilderm uth M C,Chakravarthy S,et al.Tomato Transcription factors Pti4,Pti5 and Pti6 activate De-lense responses when expressed in Arabidopsis.Plant Cell,2002,14(4):817-831.
11 Jack k Okamur O,CASTER B,Vilhrrol It,et al.The AP2 domian of APETALA2 defines alarge new family of DNA binding protein in Ambidopsis.PNAS,1997,94(6):7076-7081.
12 Shen Y G,Yah D Q,Zhang W K,et al.Novel Halophyte EREBP/AP2-type DNA Binding Protein Improves salt tolerance in Transgenic Tobacco.Aeta Botanica Sinica,2003,45(1):82-87.
13 QinF,Li Jie,ZhangG Y,et al.Isolation and Structural Analysis of DRE-Binding Transcription Factor from Maize(ZeamaysL).ActaBotanicaSinica,2003,45(3):33 1-339.
14 M asam Ohme-Taha gi and Mideaki Shinshi.Ethylene-inducible DNA binding proteins that interact with an Ethylene-responsive element.Plant CeII,1995,7(2):173-182.
15 Chen JQ,DongYi,WangY J,eta l.An AP2/EREBP type transcription factor gene from riceis coldinducible and encodes a nuclear ocalized protein.Theor Appl Genet,2003,107:972-979.
16 Susan Y,Fujinmoto,MasaruOhta,et al.Arabidopsis ethylene-responsive element binding Factors act as transcriptional activators or repressors of GCC box-mediated gene expression.Plant Cell.2000,12(3):393～404.
17 NakanoT,Suzuki K,FujimuraT,and ShiushiH.Genome-wide analysis of the ERF gene family in Arabidopsis and rice.Plant Physiology,2006,140:141～432.
18 Huang Bo,Jin LongGuo,Liu Jin Yuan.Molecular cloning and functional characterization of a DREB-1/CBF-like gene (GhDREBIL) from cotton.Sci China C Life Sci,2007,50 (1):7～14.
19 Latini A,Rasi C,Sperandei M,et al.Identification of a DREB-related gene in Triticum durum and its expression under water stress conditions.Annals of Applied Biology,2007,150 (2):187～195.
20 Dubouzet J G,Sakuma Y,Ito Y,et al.Os2DREB genes in rice,Oryza sativa L.,encode transcription acti vators that function in drought,high-salt and cold responsive gene expression.Plant Journal,2003,33 4:751～763.
21 Ernst HA,Olsen AN,Skriver K,et al.Structure of the conserved domain of ANAC,a member of the NAC family of transcription factors.EMBO Rep,2004,5 (3):297～303.
22 Cohen SX,Moulin M,Hashemolhosseini S,et al.Structure of the GCM domain-DNA complex:a DNA-binding domain with a novel fold and mode of target site recognition.EMBO J,2003,22:1835～1845.
23 Addie Nina Olsen,Heidi A Ernst,Leila Lo Leggio and Karen Skriver.NAC transcription factors:structurally distinct,functionally diverse.Trends in Plant Science,2005,10(2):79～87.
24 Liu LS,White MJ,MacRae TH.Transcription factors and their genes in higher plants.European Journal of Biochemistry,1999,262 (2):247～257.
25 Ooka H,Satok K,Doi K,et al.Comprehensive analysis of NAC family genes in Oryza sativa and Arabidopsis thaliana.DNA Research,2003,10 (6):239～247.
26 Fang Y,You J,Xie K,et al.Systematic sequence analysis and identification of tissue-specific or stress-responsive genes of NAC transcription factor family in rice.Mol Genet Genomics,2008,280(6):547-63.
27 Ren T,Qu F,Morris TJ.HRT Gene Function Requires Interaction between a NAC Protein and Viral Capsid Protein to Confer Resistance to Turnip Crinkle Virus.Plant Cell,2000,12 (10):1917～1925.
28 Cour T,Kiemer L,Molgaard A,et al.Analysis and prediction of leucine-rich nuclear export signals.Protein Engineering,Design & Selection,2004,17 (6):527～536.
29 Tran LSP,Nakashima K,SakumaY,et al.Isolation and Functional Analysis of Arabidopsis Stress-Induc ible NAC Transcription Factors That Bind to a Drought-Responsive cis-Element in the early responsive to dehydration stress 1 Promoter.Plant Celh 2004,16(9):2481-2498.
30 F(?)bio T S,Nogueira,Paulo S,et el.SsNAC23,a member of the NAC domain protein family,is associated with cold,berbivory and water stress in sugarcane.Plant Science,2005,169(1):93-106.
32 Tadashi Kunieda,Nobutaka Mitsuda,Masaru OhmeTakagi,et al.NAC Family Proteins NARS1/NAC2and NARS2/NAM in the Outer Integument Regulate Embryogenesis in Arabidopsis.Plant Cell,2008,20(10):2631-2642.
33 Mitsuds N,Iwase A,Yamamoto H,et el.NAC Transcription Factors,NST1 and NST3,Are Key Regulators of the Formation of Secondary Walls in Woody Tissues of Arabidopsis.Plant Cell,2007,19:270-280.
34 Mitsuda N,Seki M,Shinozaki K,et al.The NAC Transcription Factors NST1 andNST2 of Arabidopsis Regulate Secondary Wall Thickenings andAre Required for Anther Dehiscence.Plant Cell,2005,17:2993 -3006.
35 Zhung R,Richardson E A,Ye Z.-H.Two NAC domain transcription factors,SND1 and NST1,function redundantly in regulation of secondary wall synthesis in fibers of Arabidopsis.Planta,2007,225:160-3-1611.
36 路子显,常团结,刘翔等.植物碱性亮氨酸拉链(bZIP)蛋白研究进展[J].遗传.2001,23(6).564-570.
37 Nakagawa H,Ohmiya K,Hattori T,et el.A rice bZIP protein,designated OSBZS,is rapidly induced by absciscic[J].The Plant Journal,1996,9:217-227.
38 Riechmann JL,Heard J,Martin G,et el.Arabidopsis transcription factors:genome-wide comparative analysis among Eukaryotes.Science,2000,290:2105-2110.
39 Lipsick JS.One billion years of MybOncogene,1996,13(2):223-235.
40 SM,Gilson E.The telobox,a Myb-relatedtelomeric DNA binding motif found in proteins from yeast,plants and human.Nuclaeic Acids Res,1996,24:1294-1303.
41 Krnaz HD,Denkeamp M,Greco R,et al.Towards functional characterization of members of the R2R3-M YB gene family frim Arabidopsis thaliana.The Plant Jiurnal,1998,16:263-276.
42 Ite M.Factors controlling cyclin Bexpression.Plant Mol Biol,2000,43:677-690.
43 Krnaz H,Seholz K,WeisshsarB.c-MYB oncogene-like genes encoding three MYB repeats occur in all major plant lineages.The Plant Journal,2000,21:231-235.
44 Eulgem T,Rushton PJ,Robatzek S,etal.The WRKY superfamily of plant transcription factors.Trends Plant Sci,2000,5(5):199-206.
45 Dong J,Chen C,Chen Z.Expression profiles of the Arabidopsis WRKY gene superfamily during plant defense response [J].PlantMol Biol,2002,51:21～37.
46 Qiu Y,Jing S,Fu J,et al.Cloning and analysis of expression profileof 13 WRKY genes in rice [J].Chin Sci Bull,2004,49:2159～2168.
47 EulgemT,RushtonPJ,SchmelzerE,etal.Early nuclear events in plant defence signaling:rapid gene activation by WRKY transcription factors [J].The EMBO J,1999,18:4689～4699.
48 Yang P,Wang Z,FanB,et al.Apathogen and salicylic acid-induced WRKYDNA-binding activity recognizes the elicitor response element of the tobacco class Ichitinase genepromoter [J].Plant J,1999,18:141～149.
49 Yu D,Chen C,Chen Z.Evidence for an important role of WRKYDNA binding protein in the regulation of NPR1 gene expression [J].The Plant Cell,2001,13:1527～1539.
50 DONGJ X,CHEN CH H,CHEN ZH X.Expression profiles of the Arabidopsis WRKY gene superfamily during plant defense response [J].PlantMol.Biol.,2003,51 (1):21～37.
51 ZHANG ZH L,XIE ZH,ZOU XL,et al.A rice WR KY gene encodes a transcriptional repressor of the gibberellin signaling pathway in aleurone cells[J].PlantPhysiol,2004,134(4):1500～1513.
52 Liming Xiong,Jian Kang Zhu.Cell signaling during cold,drought and salt stress.Plant Cell,2002:S165～S183.
53 Xiong L,Itshitani M,Lee H,Zhu J K.The Arabidopsis LOS5/ABA3 locus encodes a molybdenum cofa-ctor sulfurase and modulates cold and osmotic stress-responsive gene expression.Plant Cell,2001,13:2063～2083.
54 Shinozaki K,Seki M,Regulatory network of gene exp ression in the drought and cold stress respons es.Current Opinion of Plant Biology,2003,6:410～417.
55 CheongYH,et al.Transcriptional profiling reveals novel interactions between wounding,pathogen,abiotic stress,and hormonal responses in Arabidopsis.Plant Physiology,2002,125:661～677.
56 Dwayne Hegedus,Min Yu,Doug Baldwin,et al.Molecular characterization of Brassicanapus NAC domain transcriptional activators induced in response to biotic and abiotic stress.Plant Molecular Biolog-y,2003,53 (3):383～397.
57 Chen WQ,Glaze brook J,et al.Expression profile matrix of Arabidopsis transcription factor genes suggests their putative functions in response to environmental stresses.PlantCell,2002,14:559～574.
58 Zhu T,Provart N.J.Transcriptional responses to low temperature and their regulation in Arabidopsis.Canadian Journal of Botany,2003,81:1168～1174.
59 Riechmann JL,Heard J,Martin G,et al.Arabidopsis Transcription Factors:Genome-WComparative Analysis Among Eukar-yotes.Science,2000,15,290(5499):2105-10.
60 Hirt H.MAP kinases in plant signal transduction.Res Prob Cell Dilt,2000,27:1-9.
61 田云,卢向阳,彭丽莎,方俊.植物WRKY转录因子结构特点及其生物学功能.遗传,2006,28(12):1607-1612.
62 Xiong L,Schurnaker K,and Zhu JK.Cell signaling during cold,drought and salt stress.Plant Cell,2002,14:5165-183.
63 Knight H.Calcium signaling during abiotic stress in plants.Int Rev Cytol,2000,195:269-325.
64 Cheng S H,Wilmnann M R,Chen H C,Sheen J,Calcium signaling through protein kinases:the Arabidopsis calcium-dependent protein kinase gene family.Plant Physiol,2002,29:469-485.
65 Hatmon A C,Gribskov M,Harper J E.CDPKs-a kinase for every Ca~(2+) signal?.Trends Plant Sci,2000,5:154-159.
66 Chico J M,Ralces M,Tellezinon M T,et al.A calcium-dependent protein kinase is systemically included upon wounding in tomato plants.Plant Phvsiol,2002,128:256-70.
67 Jeong HJ,JwaNS,Kim KN.Identification and characterization of protein kinases that int-eract with the CBL3 calcium sensor in Arabidopsis.Plant Sci,2005,169:1125-1135.
68 Qiu QS,Guo Y,Dietrich M,et al.Regulation of SOSl,a plasma membrane Na+/H+exchanger in Arabidopsis thaliana,by SOS2 and SOS3.ProcNatl Acad Sci USA,Z002,99:8436-8441.
69 Shinozaki K and Yamagnchi-Shinozak K.Gene networks involved in stress drought response and toleranc [J].Journal of Experimental Botany,2007,58:221-227.
70 Kasuga M,Liu Q,Miura S,et al.Improving drought,salt and freezing tolerance by gene transfer of a single stress-inducible transcription factor.Nature Biotechnology,1999,17:287-281.
71 Pellegrineschi A.,Reynolds M.,PachecoM.Stress-induced expression in wheat of the Arabidopsis thaliana DREB1 Agene delays water stress under greenhouse conditions.Genome,2004,47(3):493-500.
72 ItoY.,Katsura K.,Maruyama K.,etal.Functional Analysis of Rice DREB1/CBF-Type transcription factors Involved in Cold-Responsive Gene Expression in Transgenic Rice.Plant Cell Physiol,2006,47:141-153.
73 高世庆,徐慧君,程宪国等.科学通报,2005,50(23):2617-2625.
74 Kazuo Nakashima,Lain-Son P,Tran,et al.Functional analysis of a NAC-type transcription factor OsNA -C6 involved in abiotic and biotic stress-responsive gene expression in rice.Plant J,2007,51(4):617-630.
75 Honghong Hu,Jun You,Yujie Fang,et al.Characterization of transcription factor gene SNAC2 conferring cold and salt tolerance in rice.Plant Mol Biol,2008,67(1-2):169-181.
76 Naoki Yokotani,Takanari Ichikawa,Youichi Kondou,et al.Tolerance to various environmental stresses conferred by the salt-responsive rice gene ONAC063 in transgenic Arabidopsis.Planta 2009,229(5):1065-75.
77 Hwang E W,Kim K A,Park S C,et al.Expression profiles of hot pepper(Capsicum annum) gene under cold stress conditions.Biosci,2005,30(5):657-667.
78 Hongying Duan,Fuguang Li,Xudong Wu,et al.Cloning and characterization of two EREBP transcripting factors from cotton(Gossypium hirsutum L.)[J].Biochemistry(Moscow),2006,71(3):285-293.
79 柳展基,邵凤霞,唐桂英,单雷,毕玉平.一个新的玉米NAC类基因(ZmNAC1)的克隆与分析.遗传,2009,31(2):199-205.
80 Chen Ming,Wang Qiao-Yan,Cheng Xian-Guo,Xu Zhao-Shi.GmDREB2,a soybean DRE-binding transcription factor,conferred drought and high-salt tolerance in transgenic plants.Biochem Biophys Res Commun,2007,353(2):299-305.
81 Liu N,Zhong NQ,Wang GL,et al.Cloning and functional characterization of PpDBF1 gene encoding a DRE-binding transcription factor from Physcomitrella patens.Planta,2007,226(4):827-38.
82 Hu H,Dai M,Yao J,et al.Overexpressing a NAM,ATAF,and C U C(NAC) transcription factor enhances drought resistance and salt tolerance in rice.Proc Natl Acad Sci USA,2006,103(35):12987-12992.