海岛棉DREB基因克隆及其在转基因烟草中的功能验证
|
摘要
DREB(干旱响应元件结合蛋白)转录因子是近十年来新发现的在植物生长发育、激素调节和抵抗逆境等方面发挥重要作用的植物特异性转录因子。在逆境胁迫下,它通过与DRE元件(脱水应答元件)特异性结合,从而激活许多逆境诱导基因的表达,提高植物对逆境的抵抗和忍耐力,使植物的抗逆性得到综合性改良,是迄今为止最理想的植物抗逆工程基因。
本研究通过同源克隆的方法从海岛棉中克隆了一个DREB转录因子基因,然后将其转入烟草,对它的基本特性进行了研究,证实了这个基因具有DREB类转录因子的基本特性,它在转基因烟草中的过量表达可以提高烟草抵抗非生物胁迫的能力。主要研究结果如下:
1.根据已发表的陆地棉GhDREB1基因序列设计特异性引物P1和P2,进行RT-PCR扩增,得到一个长651bp,编码216个氨基酸的基因,命名为XHDREB。同源性分析表明,XHDREB与陆地GhDREB1,拟南芥CBF1/DREB1B和DREB1A/CBF3在氨基酸水平上的序列相同性分别为85.19%,47.32%和46.64%。
2.分析XHDREB基因编码的氨基酸序列,其相对分子量为24.6 kDa,等电点为9.46,它具备DREB1转录因子的特征:含有58个氨基酸组成的AP2 DNA结合域以及两个DREB1/CBF特征氨基酸序列PKKRAGRKKFRETR和DSAWR。
3.设计含有KpnⅠ和PstⅠ酶切位点的特异性引物DR-1和DR-2,用KpnⅠ和PstⅠ分别对XHDREB基因和pCAMBIA2300-35SOCS质粒进行双酶切,用T4 DNA Ligase连接,构建植物表达载体pC-XHDREB。
4.利用农杆菌介导法转化烟草,通过Kan抗性、PCR及RT-PCR筛选鉴定阳性烟草。对转基因烟草进行干旱、高盐等抗逆性分析并且对其相关生理生化指标进行测定,结果显示转基因烟草对这些非生物胁迫均有一定的抵抗能力。
3.利用荧光定量RT-PCR对XHDREB在转基因烟草中的表达情况进行分析。结果显示:XHDREB在干旱胁迫下的表达情况与陆地棉GhDREB1的表达情况有所不同,但与拟南芥以及其他植物中DREB1/CBF类基因的相似,在高盐胁迫下的表达情况与陆地棉GhDREB1、拟南芥以及其他植物中DREB1/CBF类基因的相似。但是在低温胁迫下的表达情况与陆地棉GhDREB1、拟南芥以及其他植物中DREB1/CBF类基因有一定差异。
DREB(dehydration responsive element binding protein)transcription factors are a family of functionally diverse proteins.They are unique to plants and play an important role in regulation of plant growth and development,homone reguation and responses to various stresses.The DREB transcription factor can specifically combine with the DRE(dehydration responsion element)and activate expression of many stress-induced genes under the stress and increasing the capacity of resistance and endurance to adversity of plant,is the best adversity resistance gene in plant by far.
In this study,homology clonging methods in the new cloned from island cotton,a DREB transcription factor genes into tobacco by its,its basic characteristics and biological functions have been studied and proved than genes DREBs of transcription factors basic characteristics,which in transgenic tobacco plants over-expression can improve the capacity of resistance to abiotic stress.The major findings are as follows:
1.According to published gene sequence of land cotton GhDREB1 transcription factor,specific primers P1 and P2 were designed and a gene with 651bp in length were amplified by RT-PCR.The gene contained a complete sequence encoding 216 amino acids,named XHDREB.Homological analysis showed that 85.19% amino acids in the sequences were similar to cotton GhDREB1 gene,47.32% amino acids in the sequences were similar to AtCBF1/DREB1B and 46.64% amino.acids in the sequences were similar to AtDREB1A/CBF3.
2.Analysis indicated that the deduced relative molecular weight of the amino acid was 24.6 kDa for XHDREB.It contains the typical characteristics to DREB1 transcription factor:58 amino acids constitute a DNA binding region AP2 and two typical characteristics amino acids sequence to DREB1/CBF:PKKRAGRKKFRETR and DSAWR.
3.The sensed specific primer DR-1 and DR-2,containing KpnⅠand PstⅠrestriction sites,were designed.Plasmid containing XHDREB gene and pCAMBIA2300-35SOCS vector was digested with KpnⅠand PstⅠ,respectively.The T4DNA ligase was used to be ligated,sensed plant expression vector pC-XHDREB was built.
4.Transforming tobacco by using agrobacterium-mediated.Screening and identification of positive tobacco by Kan resistance,PCR and RT-PCR.The drought,high salt resistance and other resistance of transgenic tobacco were analysised.And the biochemical and physiology quota were measured,the results indicate that the transgenic tobacco had some of resistance ability to these abiotic stress.
5.The expression of XHDREB in transgenic tobacco were analyzed by using real time quantitative RT-PCR.There is difference on expression levels of XHDREB under drought stress between upland GhDREB1 and island cotton,But the expression levels of XHDREB in island cotton is similar to the DREB1/CBF genes of arabidopsis and other plant.The expression levels of XHDREB under high salt stress in island cotton is similar to upland GhDREB1,arabidopsis and other plants.But the expression levels of XHDREB under cold stress in island cotton is different from upland GhDREB1,arabidopsis and other plant.
本研究通过同源克隆的方法从海岛棉中克隆了一个DREB转录因子基因,然后将其转入烟草,对它的基本特性进行了研究,证实了这个基因具有DREB类转录因子的基本特性,它在转基因烟草中的过量表达可以提高烟草抵抗非生物胁迫的能力。主要研究结果如下:
1.根据已发表的陆地棉GhDREB1基因序列设计特异性引物P1和P2,进行RT-PCR扩增,得到一个长651bp,编码216个氨基酸的基因,命名为XHDREB。同源性分析表明,XHDREB与陆地GhDREB1,拟南芥CBF1/DREB1B和DREB1A/CBF3在氨基酸水平上的序列相同性分别为85.19%,47.32%和46.64%。
2.分析XHDREB基因编码的氨基酸序列,其相对分子量为24.6 kDa,等电点为9.46,它具备DREB1转录因子的特征:含有58个氨基酸组成的AP2 DNA结合域以及两个DREB1/CBF特征氨基酸序列PKKRAGRKKFRETR和DSAWR。
3.设计含有KpnⅠ和PstⅠ酶切位点的特异性引物DR-1和DR-2,用KpnⅠ和PstⅠ分别对XHDREB基因和pCAMBIA2300-35SOCS质粒进行双酶切,用T4 DNA Ligase连接,构建植物表达载体pC-XHDREB。
4.利用农杆菌介导法转化烟草,通过Kan抗性、PCR及RT-PCR筛选鉴定阳性烟草。对转基因烟草进行干旱、高盐等抗逆性分析并且对其相关生理生化指标进行测定,结果显示转基因烟草对这些非生物胁迫均有一定的抵抗能力。
3.利用荧光定量RT-PCR对XHDREB在转基因烟草中的表达情况进行分析。结果显示:XHDREB在干旱胁迫下的表达情况与陆地棉GhDREB1的表达情况有所不同,但与拟南芥以及其他植物中DREB1/CBF类基因的相似,在高盐胁迫下的表达情况与陆地棉GhDREB1、拟南芥以及其他植物中DREB1/CBF类基因的相似。但是在低温胁迫下的表达情况与陆地棉GhDREB1、拟南芥以及其他植物中DREB1/CBF类基因有一定差异。
DREB(dehydration responsive element binding protein)transcription factors are a family of functionally diverse proteins.They are unique to plants and play an important role in regulation of plant growth and development,homone reguation and responses to various stresses.The DREB transcription factor can specifically combine with the DRE(dehydration responsion element)and activate expression of many stress-induced genes under the stress and increasing the capacity of resistance and endurance to adversity of plant,is the best adversity resistance gene in plant by far.
In this study,homology clonging methods in the new cloned from island cotton,a DREB transcription factor genes into tobacco by its,its basic characteristics and biological functions have been studied and proved than genes DREBs of transcription factors basic characteristics,which in transgenic tobacco plants over-expression can improve the capacity of resistance to abiotic stress.The major findings are as follows:
1.According to published gene sequence of land cotton GhDREB1 transcription factor,specific primers P1 and P2 were designed and a gene with 651bp in length were amplified by RT-PCR.The gene contained a complete sequence encoding 216 amino acids,named XHDREB.Homological analysis showed that 85.19% amino acids in the sequences were similar to cotton GhDREB1 gene,47.32% amino acids in the sequences were similar to AtCBF1/DREB1B and 46.64% amino.acids in the sequences were similar to AtDREB1A/CBF3.
2.Analysis indicated that the deduced relative molecular weight of the amino acid was 24.6 kDa for XHDREB.It contains the typical characteristics to DREB1 transcription factor:58 amino acids constitute a DNA binding region AP2 and two typical characteristics amino acids sequence to DREB1/CBF:PKKRAGRKKFRETR and DSAWR.
3.The sensed specific primer DR-1 and DR-2,containing KpnⅠand PstⅠrestriction sites,were designed.Plasmid containing XHDREB gene and pCAMBIA2300-35SOCS vector was digested with KpnⅠand PstⅠ,respectively.The T4DNA ligase was used to be ligated,sensed plant expression vector pC-XHDREB was built.
4.Transforming tobacco by using agrobacterium-mediated.Screening and identification of positive tobacco by Kan resistance,PCR and RT-PCR.The drought,high salt resistance and other resistance of transgenic tobacco were analysised.And the biochemical and physiology quota were measured,the results indicate that the transgenic tobacco had some of resistance ability to these abiotic stress.
5.The expression of XHDREB in transgenic tobacco were analyzed by using real time quantitative RT-PCR.There is difference on expression levels of XHDREB under drought stress between upland GhDREB1 and island cotton,But the expression levels of XHDREB in island cotton is similar to the DREB1/CBF genes of arabidopsis and other plant.The expression levels of XHDREB under high salt stress in island cotton is similar to upland GhDREB1,arabidopsis and other plants.But the expression levels of XHDREB under cold stress in island cotton is different from upland GhDREB1,arabidopsis and other plant.
引文
[1] Yamaguchi-Shinozaki K,Shinozaki K.A novel cis-acting element in an Arabidopsis gene is involved in responsiveness to drought,low-temperature or hight-salt stress[J].Plant Cell,1994(6):251-264.
[2] Dubouzet J,Sakuma Y,et al.OsDREB genes in rice,Oryza sativa L.,encode transcription activators that function in drought- , high-salt-and cold-responsive gene expression[J].Plant Cell.2003(13):751-763.
[3]刘强,张贵友,陈受宜.植物转录因子的结构与调控作用[J].中国科学,2000(4):1465-1474.
[4] Riechmann L,Heard J.Arabidpsis Transcription Factors:Genome-Wide Comparative Analysis Among Eukaryotes[J].Science.2000(9):2105-2110.
[5] Yamaguchi-Shinozaki K,Shinozaki K.Transcriptional regulatory networks in cellular responseand the tolerance to dehydration and cold stresses[J].Annu Rev Plant Molbiol.2006(27):781-803.
[6] Aukerman M,Schmidt R,Burr B,Burr F A.Anarginine to lysine substitution in the bZIP domain of an Ap2 mutant in maize abolishes specific DNA binding[J].Genes Dev.1991(2):310-320.
[7] Hu Y X,Wang Y X,Liu X.Arabidopsis Ravl is down-regulated by brassin-osteroid and may act as a negative regulator during plant development[J].Cell Research.2004(14):8-15.
[8] Meshi T,Iwabuchi M.Plant transcription factors[J].Plant Cell Physiol,1995(8):1405-1420.
[9] Washburn K B,Davis E A,Ackerman S.Co-activators and tafs of transcription activation in wheat[J].Plant Mol Biol.1997(6):1037-1043.
[10] Hill A,Nantel A,Rock C D,Quatrano R S.A conserved domain of the vivi parous-gene product enhances the DNA binding activity of the bZIP protein EMBP-1 and other transcription factors[J].J.Biol.Chem.1996,271(7):66-74.
[11] Goff S A,Cone K C,Chandler V L.Functional analysis of the transcripti on a activator ecoded by the maize B gene:evidence for a direct functional interaction between two classes of regulatory proteins[J].Genes Dev.1992,6(5):64-75.
[12] Liu L,White M J,Mac Rae T H.Transcription factors and their genes in higher plants[J].Eur Biochem.1999(2):247-257.
[13] Baker S S,Wilhelm K S,Thomasshow M E.The 5’-region of Arabdopsis tha liana corl5a has cis-acting elements that confer cold-,drought-and ABA-regulated gene expression[J].Plant Mol Biol.1994(24):701-713.
[14] Shinozaki K,Yamaguchi-Shinozaki K.Gene expression and signal transduction in water-stress response[J].Plant Physiol.1997(15):327-333.
[15] Busk P K,Jensen A B,Pages M.Regulatory elements in vivo in the promoter of the abscisic acid responsive gene rab17 from maize[J].Plant Cell.1997(11):1285-1295.
[16] Sakuma Y,Liu Q,Dubouzel J G,Abe H.DNA-Binding Specificity of the ERF/AP2 Doma-in of Arabidopsis DREBs,Transcription factors involved in dehydration-and cold-inducible g-ene[J].Biochem Biophysres Commun.2002(29):998-1009.
[17] Okamuro K,Caster B,Villarroel R.The AP2 domain of AP2/EREBP define a large new family of DNA binding Protein in Arabidopsis[J].P Natl Acad Sci Usa.1997,94(13):707-708.
[18] Allen M D,Yamasaki K,Ohme-Takagi M,Tateno M,Suzuki M.A novel modle of DNA recognition by a beta-sheet revealed by the solution structure of the GCC-box binding domain in complex with DNA[J].Embo J.1998(17):5484-5496.
[19] Shinozaki K,Yamaguchi S K.Molecular responses to dehydration and low-temperature:differenceand cross-talk between two stress signaling pathways[J].Curr Opin Plant Bio.2000(3):217-223.
[20] Kizis D,Lumbreras V,Pages M.Role of AP2/EREBP transcription factors in gene regulaton during abiotic stress[J].Plant Cell.2001(8):187-189.
[21] Stockinger E,Gilmour S,Thomashow M E.Arabidopsis thaliana CBF1 encodes an AP2 domain containing transcription activator that binds to the C-repeat/DRE,a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit[J].P Natl Acad Sci Usa.1997(9):1035-1040.
[22] Lin Q,Kasuga M,Sakuma Y,Abe H,Miura S,Yamaguchi-Shinozaki K,Shinozaki K.Two transcription factors,DREB1 and DREB2,with an EREBP/AP2 DNA-binding domain separate two cellular signal transduction pathways in drought-and low-temperature-responsivegene expression in Arabidopsis[J].Plant Cell.1998(10):1391-1406.
[23] Riechmann J L,Heard J,Martin G,Reuber L,Jiang C,Keddie J,Adam L,Pineda O,Ratcliffe O J,Samaha R R,Creelman R,Pilgrim M,Broun P,Zhang J Z,Ghandehari D,Sherman B K,Yu G.Arabidopsis transcription factors:genome-wide comparative analysis am-ong eukaryotes[J].Science.2000(29):2105-2110.
[24] Wei G,Pan Y,Lei J,Zhu Y X.Molecular cloning,phyogenetic analysic expressional profiling and in vitro studies of TINY2 from Arabidopsis thaiana[J].Biochem Mol Biol.2005(38):440-446.
[25] Chen M,Wang Q Y,Cheng X G,et al.GmDREB2,a soybean DRE-binding transcription factor,conferred drought and high-salt tolerance in transgenic plants[J].Biochem Biophysres Commun.2007(35):299-305.
[26] Huang B,Liu J Y.A cotton dehydration responsive element binding protein functions as a transcriptional repressor of DRE mediated gene expression[J].Biochem Biophysres Commun.2006(34):1023-1031.
[27] Kizis D,Pages M.Maize DRE-binding proteins DBF1 and DBF2 are involved in rab17 reg-ulation through the drought responsive element in an ABA-dependent pathway[J].Plant.2002(30):679-689.
[28] Koussevitzky S,Nott A.Multiple signals from damaged chloroplasts converge on a common pathway to regulate nuclear gene expression[J].Science.2007(30):79-89.
[29] Wilson K,Long D,Swinburne J,Coupland G.A dissociation insertion causes a Semi-dominant mutation that increases expression of TINY,an Arabidopsis gene related to AP2/EREBP[J].Plant Cell.1996,8(4):659-671.
[30] Gu Y Q,Wildermuth M C and Chakravarthy S.Tomato transcription factors Pti4,Pti5 and Pti6 activate defense responses when expressed in Arabodonsis[J].Plant Cell.2002(14):813-817.
[31] Karam B S,Rhonda C F and Lius Q S.Transcription factor in plant defense and stress res-ponse[J].Cell Signaling and Gene Regulation.2002(5):430-439.
[32] Hong Z.Removal of feed back inhibition of aply roling-5-carboxylate synthase results increa-sed praline accumulation and protection of plants from osmotic stress[J].Plant physiol.2000(12):1129-1136.
[33] Thomashow F.Plant cold acclimation:Freezing tolerance genes and regulatory mechanisms[J].Annu Rev Plant Physiol Plant Mol Biol.1999(15):571-599.
[34] Hasegawa M,Bressan R A,Zhu J K,Bohnert H J.Plant cellular and molecular responsive to high salinity[J].Annu Rev Plant Physiol Plant Mol Biol.2000(51):463-499.
[35] Xiong L M,Zhu J K.Regulation of abscisic acid biosynthesis[J].Plant Physiol.2003,133:29-36.
[36] Yamaguchi-Shinozaki K,Kasuga M,Liu Q,Nakashima K,Sakuma Y,Abe H,Shinwariz K,Scki M,Shinozaki K.Biological mechanisms of drought stress response[J].Plant Physiol.2002(23):1-8.
[37] Shinozaki K,Yamaguchi-Shinozaki K,Seki M.Regulatory network of gene expression in thedrought and cold stress responses[J].Plant Mol Biol.2003(6):410-417.
[38] Verslues P E,Zhu J K.Before and beyond ABA:upstream sensing and internal signals that determine ABA accumulation and response under abiotic stress[J].Bioche mical Society Tran-sactions.2005(33):375-37.
[39] Kim Y H,Yang K S,Ryu S H,et al.Molecular characterization of a cDNA encoding DR-E-binding transcription factor from dehydration-treated fibrous roots of sweet potato[J].Plant Physiol.2008(2):196-204.
[40] Li Qing Liu,Kai Zhu,Yan Fang,et al.Molecular cloning,expression profiling and transactivation property studies of a DREB2-like gene from chrysanthemum(Dendranthema vestitum)[J].Plant.2008,121(2):215-226.
[41] Hong J P,Kim W T.Isolation and functional characterization of the Ca-DREB1 gene encod-ing a dehydration-responsive element binding-factor-like protein in hot pepper(Capsicum ann-uuml.cv Pukang)[J].Plant.2005(22):875-888.
[42] Haake V,Cook D,Riechmann J,et al.Transcription factor CBF4 is a regulator of drought adaptation in Arabidopsis[J].Plant Physiol.2002(13):639-48.
[43] Shen Y G,Zhang W K,Yan D Q,et al.Characterization of a DRE-binding transcription factor from ahalophyte Atriplex hortensis[J].Theor Appl Genet.2003,107:155-161.
[44] Li X P,Tian A G,et al.Soybean DRE-binding transcription factors that are responsive to abiotic stresses[J].Theor Appl Genet.2005,110:1355-1362.
[45] Yang Y,Wu J,Zhu K,et al.Identification and characterization of two chrysanthemum(Dendronthemax moriforlium)DREB genes,belonging to the AP2/EREBP family[J].Mol Biol.2007(6):10-11.
[46] Jaglo K R,Kleff S,Amundsen K L,Zhang X,Haake V,Zhang J Z,Deits T,Thomashow M F.Component soft the Arabidopsis C-repeat/dehydration-responsive element binding fac-tor cold-response pathway are conserved in Brassica napus and other plant species[J].Plant Physiol.2001(127):910-917.
[47] Shen Y G,Zhang W K,He S J,et al.An EREBP/AP2-type protein in Triticum aestivum was a DRE-binding transcription factor induced by cold,dehydration and ABA stress[J].Theo-r Appl Genet.2003,106:923-930.
[48] Shen Y G,Yan G Q,Zhang W K,Du B X.Novel halophyte EREBP/AP2-Type DNA binding protein improves salt tolerance in transgenic tobacco[J].Acta Bot.Sin.1997(45):82-87.
[49] Hsieh T H,Lee J T,Charng Y Y,Chan M T.Tomato plants ectopically expressing Arabidopsis CBFI show enhanced resistance to water deficit stress[J].Plant Physiol.2002(130):618-626.
[50] Qin F,Sakuma Y,Li J,et al.Cloning and functional analysis of a novel DREB1/CBF tran-scription factor involved in cold-responsive gene expression in Zea mays[J].Plant Cell Physi-ol.2004(45):1042-1052.
[51] Pellegrineschi A,Reynods M,Pacheco M,et al.Stress induced expression in wheat of the Arabidopsis thaliana DREB1A gene delays water stress symptoms under greenhouse conditions[J].Genome.2004(47):493-500.
[52] Oh S J,Song S I,Kim Y S,et al.Arabidopsis CBF3/DREB1A and ABF3 in transgenic ri-ce increased tolerance to abiotic stress without stunting growth[J].Plant Physiol.2005,138:341-351.
[53] Kasuga M,Liu Q,Miura S,Yamaguchi-Shinozaki K,Shinozaki K.Improving plant drought,salt and freezing tolerance by gene transfer of a single stress inducible transcription factor[J].Nature Biotechnology.1999(17):287-291.
[54] Kasukabe Y,He L,Nada K,Misawa S,Ihara I,Tachibana S.Over expression of spermid-ine synthase enhances tolerance to multiple environmental stresses and up-regulates the expre-ssion of various stress-regulated genes in transgenic Arabidopsis thaliana[J].Plant Cell Physio-l.2004(45):712-722.
[55]王建革,苏晓华,张冰玉.植物抗旱研究工作中的问题与方法初探[J].中国农学通报.2003,16(5):6-11.
[56]李洁.植物转录因子与基因调控[J].生物学通报.2004,39(3):9-11.
[57]刘强,赵南明,Yamaguch-Shinozaki K,et al.DREB转录因子在提高植物抗逆性中的作用[J].科学通报.2000(45):11-16.
[58]黄荣峰,杨宇红,王学臣.植物对低温胁迫响应的分子机理[J].农业生物技术学报.2001(9):92-96.
[59] Agarwal P,Agarwal P K,Nair S,et al.Stress-inducible DREB2A transcription factor from pennisetum glaucum is a phosphoprotein and its phosphorylation negatively regulates its DNA-binding activity[J].Mol Genet Genomics.2007,277(2):189-198.
[60]王关林,万宏筠.植物基因工程原理与技术[M].北京:科学出版社,1998.
[61] Bray E A.Plant responses to water deficit[J].Trends Plant Sci,1997(2):48-54.
[62]张晗,信月芝,郭惠明等.CBF转录因子及其在植物抗冷反应中的作用[J] .核农学报.2006, 2(5):406-409.
[63]黄波,金龙国,刘进元.棉花中一个类DREB1/CBF基因(GhDREB1L)的分子克隆及其功能分析[J] .中国科学C辑生命科学.2006,36(5):390-397.
[64] Chen W,Provart N J,Glazebrook J,et al.Expression profile matrix of Arabidopsis transcr-iption factor genes suggests their putative functions in response to environmental stresses[J].Plant Cell.2002(14):559-574.
[65]邹奇主编.植物生理学实验指导[M] .中国农业出版社,北京,2000.
[66] R Munns.Comparative physiology of salt and water stress[J].Plant Cell Environ.2002,25(2):239-252.
[67]林海馨.植物冷害与细胞生理[M].厦门出版社,1994:131-132.
[68]潘瑞炽.植物生理学[M].北京高等教育出版社,2001:284-288.
[69] Stockinger E J,Gilmour S J,Thomashow M F.Arabidopsis thaliana CBF1 encodes an AP2 domain containing transcriptional activator that binds to the C-repeat/DRE,a cis-acting DNAregulatory element that stimulates transcription in response to low temperature and water de-ficit[J].P Natl Acad Sci Usa.1997,94(3):1035-1040.
[70] Jaglo K R,Kleff S,Amundsen K L,et al.Components of the Arabidopsis C-repeat/dehydr-ation-responsive element binding factor cold-response pathway are conserved in Brassica na-pus and other plant species[J].Plant Physiol.2001,127(3):910-917.
[71] Xue G P.An AP2 domain transcription factor HvCBF1 activates expression of cold-responsi-ve genes in barley through interaction with a (G/a)(C/t)CGAC motif[J].Biochim Biop-hys Acta.2002,1577(1):63-72.
[2] Dubouzet J,Sakuma Y,et al.OsDREB genes in rice,Oryza sativa L.,encode transcription activators that function in drought- , high-salt-and cold-responsive gene expression[J].Plant Cell.2003(13):751-763.
[3]刘强,张贵友,陈受宜.植物转录因子的结构与调控作用[J].中国科学,2000(4):1465-1474.
[4] Riechmann L,Heard J.Arabidpsis Transcription Factors:Genome-Wide Comparative Analysis Among Eukaryotes[J].Science.2000(9):2105-2110.
[5] Yamaguchi-Shinozaki K,Shinozaki K.Transcriptional regulatory networks in cellular responseand the tolerance to dehydration and cold stresses[J].Annu Rev Plant Molbiol.2006(27):781-803.
[6] Aukerman M,Schmidt R,Burr B,Burr F A.Anarginine to lysine substitution in the bZIP domain of an Ap2 mutant in maize abolishes specific DNA binding[J].Genes Dev.1991(2):310-320.
[7] Hu Y X,Wang Y X,Liu X.Arabidopsis Ravl is down-regulated by brassin-osteroid and may act as a negative regulator during plant development[J].Cell Research.2004(14):8-15.
[8] Meshi T,Iwabuchi M.Plant transcription factors[J].Plant Cell Physiol,1995(8):1405-1420.
[9] Washburn K B,Davis E A,Ackerman S.Co-activators and tafs of transcription activation in wheat[J].Plant Mol Biol.1997(6):1037-1043.
[10] Hill A,Nantel A,Rock C D,Quatrano R S.A conserved domain of the vivi parous-gene product enhances the DNA binding activity of the bZIP protein EMBP-1 and other transcription factors[J].J.Biol.Chem.1996,271(7):66-74.
[11] Goff S A,Cone K C,Chandler V L.Functional analysis of the transcripti on a activator ecoded by the maize B gene:evidence for a direct functional interaction between two classes of regulatory proteins[J].Genes Dev.1992,6(5):64-75.
[12] Liu L,White M J,Mac Rae T H.Transcription factors and their genes in higher plants[J].Eur Biochem.1999(2):247-257.
[13] Baker S S,Wilhelm K S,Thomasshow M E.The 5’-region of Arabdopsis tha liana corl5a has cis-acting elements that confer cold-,drought-and ABA-regulated gene expression[J].Plant Mol Biol.1994(24):701-713.
[14] Shinozaki K,Yamaguchi-Shinozaki K.Gene expression and signal transduction in water-stress response[J].Plant Physiol.1997(15):327-333.
[15] Busk P K,Jensen A B,Pages M.Regulatory elements in vivo in the promoter of the abscisic acid responsive gene rab17 from maize[J].Plant Cell.1997(11):1285-1295.
[16] Sakuma Y,Liu Q,Dubouzel J G,Abe H.DNA-Binding Specificity of the ERF/AP2 Doma-in of Arabidopsis DREBs,Transcription factors involved in dehydration-and cold-inducible g-ene[J].Biochem Biophysres Commun.2002(29):998-1009.
[17] Okamuro K,Caster B,Villarroel R.The AP2 domain of AP2/EREBP define a large new family of DNA binding Protein in Arabidopsis[J].P Natl Acad Sci Usa.1997,94(13):707-708.
[18] Allen M D,Yamasaki K,Ohme-Takagi M,Tateno M,Suzuki M.A novel modle of DNA recognition by a beta-sheet revealed by the solution structure of the GCC-box binding domain in complex with DNA[J].Embo J.1998(17):5484-5496.
[19] Shinozaki K,Yamaguchi S K.Molecular responses to dehydration and low-temperature:differenceand cross-talk between two stress signaling pathways[J].Curr Opin Plant Bio.2000(3):217-223.
[20] Kizis D,Lumbreras V,Pages M.Role of AP2/EREBP transcription factors in gene regulaton during abiotic stress[J].Plant Cell.2001(8):187-189.
[21] Stockinger E,Gilmour S,Thomashow M E.Arabidopsis thaliana CBF1 encodes an AP2 domain containing transcription activator that binds to the C-repeat/DRE,a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit[J].P Natl Acad Sci Usa.1997(9):1035-1040.
[22] Lin Q,Kasuga M,Sakuma Y,Abe H,Miura S,Yamaguchi-Shinozaki K,Shinozaki K.Two transcription factors,DREB1 and DREB2,with an EREBP/AP2 DNA-binding domain separate two cellular signal transduction pathways in drought-and low-temperature-responsivegene expression in Arabidopsis[J].Plant Cell.1998(10):1391-1406.
[23] Riechmann J L,Heard J,Martin G,Reuber L,Jiang C,Keddie J,Adam L,Pineda O,Ratcliffe O J,Samaha R R,Creelman R,Pilgrim M,Broun P,Zhang J Z,Ghandehari D,Sherman B K,Yu G.Arabidopsis transcription factors:genome-wide comparative analysis am-ong eukaryotes[J].Science.2000(29):2105-2110.
[24] Wei G,Pan Y,Lei J,Zhu Y X.Molecular cloning,phyogenetic analysic expressional profiling and in vitro studies of TINY2 from Arabidopsis thaiana[J].Biochem Mol Biol.2005(38):440-446.
[25] Chen M,Wang Q Y,Cheng X G,et al.GmDREB2,a soybean DRE-binding transcription factor,conferred drought and high-salt tolerance in transgenic plants[J].Biochem Biophysres Commun.2007(35):299-305.
[26] Huang B,Liu J Y.A cotton dehydration responsive element binding protein functions as a transcriptional repressor of DRE mediated gene expression[J].Biochem Biophysres Commun.2006(34):1023-1031.
[27] Kizis D,Pages M.Maize DRE-binding proteins DBF1 and DBF2 are involved in rab17 reg-ulation through the drought responsive element in an ABA-dependent pathway[J].Plant.2002(30):679-689.
[28] Koussevitzky S,Nott A.Multiple signals from damaged chloroplasts converge on a common pathway to regulate nuclear gene expression[J].Science.2007(30):79-89.
[29] Wilson K,Long D,Swinburne J,Coupland G.A dissociation insertion causes a Semi-dominant mutation that increases expression of TINY,an Arabidopsis gene related to AP2/EREBP[J].Plant Cell.1996,8(4):659-671.
[30] Gu Y Q,Wildermuth M C and Chakravarthy S.Tomato transcription factors Pti4,Pti5 and Pti6 activate defense responses when expressed in Arabodonsis[J].Plant Cell.2002(14):813-817.
[31] Karam B S,Rhonda C F and Lius Q S.Transcription factor in plant defense and stress res-ponse[J].Cell Signaling and Gene Regulation.2002(5):430-439.
[32] Hong Z.Removal of feed back inhibition of aply roling-5-carboxylate synthase results increa-sed praline accumulation and protection of plants from osmotic stress[J].Plant physiol.2000(12):1129-1136.
[33] Thomashow F.Plant cold acclimation:Freezing tolerance genes and regulatory mechanisms[J].Annu Rev Plant Physiol Plant Mol Biol.1999(15):571-599.
[34] Hasegawa M,Bressan R A,Zhu J K,Bohnert H J.Plant cellular and molecular responsive to high salinity[J].Annu Rev Plant Physiol Plant Mol Biol.2000(51):463-499.
[35] Xiong L M,Zhu J K.Regulation of abscisic acid biosynthesis[J].Plant Physiol.2003,133:29-36.
[36] Yamaguchi-Shinozaki K,Kasuga M,Liu Q,Nakashima K,Sakuma Y,Abe H,Shinwariz K,Scki M,Shinozaki K.Biological mechanisms of drought stress response[J].Plant Physiol.2002(23):1-8.
[37] Shinozaki K,Yamaguchi-Shinozaki K,Seki M.Regulatory network of gene expression in thedrought and cold stress responses[J].Plant Mol Biol.2003(6):410-417.
[38] Verslues P E,Zhu J K.Before and beyond ABA:upstream sensing and internal signals that determine ABA accumulation and response under abiotic stress[J].Bioche mical Society Tran-sactions.2005(33):375-37.
[39] Kim Y H,Yang K S,Ryu S H,et al.Molecular characterization of a cDNA encoding DR-E-binding transcription factor from dehydration-treated fibrous roots of sweet potato[J].Plant Physiol.2008(2):196-204.
[40] Li Qing Liu,Kai Zhu,Yan Fang,et al.Molecular cloning,expression profiling and transactivation property studies of a DREB2-like gene from chrysanthemum(Dendranthema vestitum)[J].Plant.2008,121(2):215-226.
[41] Hong J P,Kim W T.Isolation and functional characterization of the Ca-DREB1 gene encod-ing a dehydration-responsive element binding-factor-like protein in hot pepper(Capsicum ann-uuml.cv Pukang)[J].Plant.2005(22):875-888.
[42] Haake V,Cook D,Riechmann J,et al.Transcription factor CBF4 is a regulator of drought adaptation in Arabidopsis[J].Plant Physiol.2002(13):639-48.
[43] Shen Y G,Zhang W K,Yan D Q,et al.Characterization of a DRE-binding transcription factor from ahalophyte Atriplex hortensis[J].Theor Appl Genet.2003,107:155-161.
[44] Li X P,Tian A G,et al.Soybean DRE-binding transcription factors that are responsive to abiotic stresses[J].Theor Appl Genet.2005,110:1355-1362.
[45] Yang Y,Wu J,Zhu K,et al.Identification and characterization of two chrysanthemum(Dendronthemax moriforlium)DREB genes,belonging to the AP2/EREBP family[J].Mol Biol.2007(6):10-11.
[46] Jaglo K R,Kleff S,Amundsen K L,Zhang X,Haake V,Zhang J Z,Deits T,Thomashow M F.Component soft the Arabidopsis C-repeat/dehydration-responsive element binding fac-tor cold-response pathway are conserved in Brassica napus and other plant species[J].Plant Physiol.2001(127):910-917.
[47] Shen Y G,Zhang W K,He S J,et al.An EREBP/AP2-type protein in Triticum aestivum was a DRE-binding transcription factor induced by cold,dehydration and ABA stress[J].Theo-r Appl Genet.2003,106:923-930.
[48] Shen Y G,Yan G Q,Zhang W K,Du B X.Novel halophyte EREBP/AP2-Type DNA binding protein improves salt tolerance in transgenic tobacco[J].Acta Bot.Sin.1997(45):82-87.
[49] Hsieh T H,Lee J T,Charng Y Y,Chan M T.Tomato plants ectopically expressing Arabidopsis CBFI show enhanced resistance to water deficit stress[J].Plant Physiol.2002(130):618-626.
[50] Qin F,Sakuma Y,Li J,et al.Cloning and functional analysis of a novel DREB1/CBF tran-scription factor involved in cold-responsive gene expression in Zea mays[J].Plant Cell Physi-ol.2004(45):1042-1052.
[51] Pellegrineschi A,Reynods M,Pacheco M,et al.Stress induced expression in wheat of the Arabidopsis thaliana DREB1A gene delays water stress symptoms under greenhouse conditions[J].Genome.2004(47):493-500.
[52] Oh S J,Song S I,Kim Y S,et al.Arabidopsis CBF3/DREB1A and ABF3 in transgenic ri-ce increased tolerance to abiotic stress without stunting growth[J].Plant Physiol.2005,138:341-351.
[53] Kasuga M,Liu Q,Miura S,Yamaguchi-Shinozaki K,Shinozaki K.Improving plant drought,salt and freezing tolerance by gene transfer of a single stress inducible transcription factor[J].Nature Biotechnology.1999(17):287-291.
[54] Kasukabe Y,He L,Nada K,Misawa S,Ihara I,Tachibana S.Over expression of spermid-ine synthase enhances tolerance to multiple environmental stresses and up-regulates the expre-ssion of various stress-regulated genes in transgenic Arabidopsis thaliana[J].Plant Cell Physio-l.2004(45):712-722.
[55]王建革,苏晓华,张冰玉.植物抗旱研究工作中的问题与方法初探[J].中国农学通报.2003,16(5):6-11.
[56]李洁.植物转录因子与基因调控[J].生物学通报.2004,39(3):9-11.
[57]刘强,赵南明,Yamaguch-Shinozaki K,et al.DREB转录因子在提高植物抗逆性中的作用[J].科学通报.2000(45):11-16.
[58]黄荣峰,杨宇红,王学臣.植物对低温胁迫响应的分子机理[J].农业生物技术学报.2001(9):92-96.
[59] Agarwal P,Agarwal P K,Nair S,et al.Stress-inducible DREB2A transcription factor from pennisetum glaucum is a phosphoprotein and its phosphorylation negatively regulates its DNA-binding activity[J].Mol Genet Genomics.2007,277(2):189-198.
[60]王关林,万宏筠.植物基因工程原理与技术[M].北京:科学出版社,1998.
[61] Bray E A.Plant responses to water deficit[J].Trends Plant Sci,1997(2):48-54.
[62]张晗,信月芝,郭惠明等.CBF转录因子及其在植物抗冷反应中的作用[J] .核农学报.2006, 2(5):406-409.
[63]黄波,金龙国,刘进元.棉花中一个类DREB1/CBF基因(GhDREB1L)的分子克隆及其功能分析[J] .中国科学C辑生命科学.2006,36(5):390-397.
[64] Chen W,Provart N J,Glazebrook J,et al.Expression profile matrix of Arabidopsis transcr-iption factor genes suggests their putative functions in response to environmental stresses[J].Plant Cell.2002(14):559-574.
[65]邹奇主编.植物生理学实验指导[M] .中国农业出版社,北京,2000.
[66] R Munns.Comparative physiology of salt and water stress[J].Plant Cell Environ.2002,25(2):239-252.
[67]林海馨.植物冷害与细胞生理[M].厦门出版社,1994:131-132.
[68]潘瑞炽.植物生理学[M].北京高等教育出版社,2001:284-288.
[69] Stockinger E J,Gilmour S J,Thomashow M F.Arabidopsis thaliana CBF1 encodes an AP2 domain containing transcriptional activator that binds to the C-repeat/DRE,a cis-acting DNAregulatory element that stimulates transcription in response to low temperature and water de-ficit[J].P Natl Acad Sci Usa.1997,94(3):1035-1040.
[70] Jaglo K R,Kleff S,Amundsen K L,et al.Components of the Arabidopsis C-repeat/dehydr-ation-responsive element binding factor cold-response pathway are conserved in Brassica na-pus and other plant species[J].Plant Physiol.2001,127(3):910-917.
[71] Xue G P.An AP2 domain transcription factor HvCBF1 activates expression of cold-responsi-ve genes in barley through interaction with a (G/a)(C/t)CGAC motif[J].Biochim Biop-hys Acta.2002,1577(1):63-72.