茶陵野生稻抗冻相关转录因子的克隆以及抗冻机理的研究
|
摘要
野生稻中蕴藏着丰富的抗病虫、抗逆、抗低温、耐干旱、耐贫瘠等抗逆优良性状基因,在现代水稻抗性育种中具有独特的作用。当今一些模式植物如拟南芥的DREB转录因子的分子生物学研究取得了良好的进展,DREB转录因子的表达受非生物逆境胁迫,如干旱、高盐、低温、冻害等逆境胁迫的诱导,在植物对逆境胁迫与激素应答反应中发挥调控作用。
本研究以茶陵野生稻为材料,针对DREB转录因子开展了研究,在低温胁迫下通过分离野生稻的总RNA并反转录成cDNA,克隆了茶陵野生稻中DREB转录因子,全长958bp,编码218个氨基酸,经BLAST及蛋白质同源性分析,该序列与水稻(Oryza sativa) DREB基因的同源性98%,与小麦(Triticum monococcum) CRT/DREB4蛋白的同源性为93%；与大麦(Hordeum vulgare)CBF2A蛋白与CBFIVc-14.1蛋白分别为93%、92%。这表明已经成功克隆了野生稻中DREB转录因子序列。
为了进一步研究DREB在不同时间低温胁迫下茶陵野生稻组织中的表达情况,以水稻基因组DNA中的内参Actin序列作为内参基因,应用RT-PCR技术对DREB转录因子的表达进行半定量分析,结果显示在不同时间的低温胁迫条件下,DREB基因的表达有组织特异性,且在根中的表达更为稳定,表达量最高。通过PCR将DREB转录因子从T载体上释放,构建至植物表达载体pWM101载体中。
通过对低温胁迫下茶陵野生稻中的各种生化酶的检测,以栽培稻作为对照,分别测定在低温不同积累时间下的过氧化氢酶、过氧化物酶、超氧化物歧化酶活性,结果发现,野生稻体内酶的活性比栽培稻要高,当胁迫累积到24小时之后,酶活性依然能保持在较高的水平,与转录因子不同时间的低温胁迫下的表达量的高低在一定程度上有相似性。
Wild rice that play a unique role in resistant breeding is rich in excellent gene such as anti-worm, anti-low temperature and drought resistance, poor resistance, and so on. Today some model plants as Arabidopsis of which DREB transcription factor has made good progress in molecular biology, DREB transcription factor expressed by induction of abiotic stress, such as drought, high salinity, low temperature, freezing and other Stress, which play a regulatory role in response to stress and hormone.
Using Chaling wild rice as material in the study to research DREB transcription factor, separated total RNA of wild rice under low temperature stress and reverse transcript it into cDNA, Sequencing analysis showed the cDNA sequence be cloned is 958bp, which can be translated into a putative 218 amino acids protein. The cones-quence of Blast showed:the sequence of rice (Oryza sativa) DREB gene homology 98%, and wheat (Triticum monococcum) CRT/DREB4 protein homology of 93%; and barley (Hordeum vulgare) CBF2A and CBFIVc-14.1 protein were 93%,92%. This indicated that DREB transcription factor sequence has been successfully cloned.
To further study the DREB expression model in tissue of Chaling wild rice under low temperature stress at different time, so as to Actin sequence of rice genome DNA sequence as an internal control, make DREB transcription factor expression semi-quantitative analysis with RT-PCR technique. The result showed DREB genes have tissue-specific expression at different time of cold stress conditions and expression in roots is more stable, the highest expression level is detected in root. DREB transcription factor is release from the T vector By PCR, and constructed to the plant expression vector pWM101 vector.
Compared with cultivated rice, the CAT (Catalase), SOD (superoxide), POD (Peroxidase dismutase) of Chaling wild rice is detected under low temperature at different time. The results found that the enzyme activity of wild rice is higher than the cultivated rice, when the stress accumulated to 24 hours the enzymes activity of wild rice is still maintained at a high level, which is similar with DREB transcription factors expression model under cold stress at different times.
本研究以茶陵野生稻为材料,针对DREB转录因子开展了研究,在低温胁迫下通过分离野生稻的总RNA并反转录成cDNA,克隆了茶陵野生稻中DREB转录因子,全长958bp,编码218个氨基酸,经BLAST及蛋白质同源性分析,该序列与水稻(Oryza sativa) DREB基因的同源性98%,与小麦(Triticum monococcum) CRT/DREB4蛋白的同源性为93%；与大麦(Hordeum vulgare)CBF2A蛋白与CBFIVc-14.1蛋白分别为93%、92%。这表明已经成功克隆了野生稻中DREB转录因子序列。
为了进一步研究DREB在不同时间低温胁迫下茶陵野生稻组织中的表达情况,以水稻基因组DNA中的内参Actin序列作为内参基因,应用RT-PCR技术对DREB转录因子的表达进行半定量分析,结果显示在不同时间的低温胁迫条件下,DREB基因的表达有组织特异性,且在根中的表达更为稳定,表达量最高。通过PCR将DREB转录因子从T载体上释放,构建至植物表达载体pWM101载体中。
通过对低温胁迫下茶陵野生稻中的各种生化酶的检测,以栽培稻作为对照,分别测定在低温不同积累时间下的过氧化氢酶、过氧化物酶、超氧化物歧化酶活性,结果发现,野生稻体内酶的活性比栽培稻要高,当胁迫累积到24小时之后,酶活性依然能保持在较高的水平,与转录因子不同时间的低温胁迫下的表达量的高低在一定程度上有相似性。
Wild rice that play a unique role in resistant breeding is rich in excellent gene such as anti-worm, anti-low temperature and drought resistance, poor resistance, and so on. Today some model plants as Arabidopsis of which DREB transcription factor has made good progress in molecular biology, DREB transcription factor expressed by induction of abiotic stress, such as drought, high salinity, low temperature, freezing and other Stress, which play a regulatory role in response to stress and hormone.
Using Chaling wild rice as material in the study to research DREB transcription factor, separated total RNA of wild rice under low temperature stress and reverse transcript it into cDNA, Sequencing analysis showed the cDNA sequence be cloned is 958bp, which can be translated into a putative 218 amino acids protein. The cones-quence of Blast showed:the sequence of rice (Oryza sativa) DREB gene homology 98%, and wheat (Triticum monococcum) CRT/DREB4 protein homology of 93%; and barley (Hordeum vulgare) CBF2A and CBFIVc-14.1 protein were 93%,92%. This indicated that DREB transcription factor sequence has been successfully cloned.
To further study the DREB expression model in tissue of Chaling wild rice under low temperature stress at different time, so as to Actin sequence of rice genome DNA sequence as an internal control, make DREB transcription factor expression semi-quantitative analysis with RT-PCR technique. The result showed DREB genes have tissue-specific expression at different time of cold stress conditions and expression in roots is more stable, the highest expression level is detected in root. DREB transcription factor is release from the T vector By PCR, and constructed to the plant expression vector pWM101 vector.
Compared with cultivated rice, the CAT (Catalase), SOD (superoxide), POD (Peroxidase dismutase) of Chaling wild rice is detected under low temperature at different time. The results found that the enzyme activity of wild rice is higher than the cultivated rice, when the stress accumulated to 24 hours the enzymes activity of wild rice is still maintained at a high level, which is similar with DREB transcription factors expression model under cold stress at different times.
引文
[1]高桥成人,角田重三郎.水稻生物学[M].武汉大学生物系遗传研究室译.武汉：武汉大学出版社,1988.
[2]傅立国.中国植物红皮书(第一册)[M].北京：科学出版社,1992,314～316
[3]史冬燕,杨明挚等.元江普通野生稻叶片cDNA文库的构建及部分基因片段分析[J].HEREDITAS(Beijing)2008,30(6):776-780
[4]鄂志国,郑传举等.中国野生稻资源及其在水稻抗性育种上的应用[J].中国稻米,2008,6(4)：3-6
[5]MERRILLED.Oryza sativaL[J].Philip Journ Sci,1917,12(2):2.
[6]范树国,张再君,刘林,等.中国野生稻的种类、地理分布及其特征特性综述[J].武汉植物学研究,2000,18(5)：417-425.
[7]燕惠民,贺益娥,湖南野生稻资源调查及其保护措施[J].种子,2007,1(26)：88-90
[8]钟代彬,罗利军,应存山.野生稻在栽培稻育种中的应用[J].种子,1995,(1)：25～29.
[9]江川,王金英,李清华.野生稻的优异特性及其在水稻育种中的利用[J].福建稻麦科技,2003,21(3)：8～10.
[10]彭绍裘,魏子生,毛昌祥,等.云南省疣粒野生稻、药用野生稻和普通野生稻多抗性鉴定.植物病理学报,1982,12(4)：58-60.
[10]章琦.普通野生稻抗白叶枯病新基因被正式命名为Xa-23[J].作物杂志,2003,(1)：20-20.
[11]潘海军,王春连,赵开军.水稻抗白叶枯病基Xa-23的PCR分子标记定位及辅助选择[J].作物学报,2003,29(4)：501-507.
[12]姜文正,涂英文,丁忠华,等.东乡野生稻研究[J].作物品种资源,988,(3)：1-4.
[13]徐羡明,曾列先,林壁润,等.普通野生稻种质资源对纹枯病的抗性鉴定.植物病理学报,1992,22(4)：300
[14]徐羡明,林壁润,曾列先,等.普通野生稻种质资源对细菌性条斑病的抗性鉴定.植物保护,1991,17(6)：4-5.
[15]覃瑞,魏文辉,宁顺斌,等.利用水稻BAC克隆对Gm-2和Gm-6在药用野生稻中的FISH定位[J].中国农业科学,2001,34(1)：1-4
[16]Ishii T,Brar DS,Multani DS,Khush GS.Molecular tagging of genes for brown planthopper resistance and earliness introgressed from Oryza australiensis into cultivated rice,O.sativa.Genome,1994,37(2):217-221.
[17]李青,罗善昱,黄润清,等.广西药用野生稻抗褐稻虱研究简报.广西植保,1988,15(3)：165-166.
[18]李荣柏,秦学毅,韦素美等.普通野生稻稻褐飞虱抗性在水稻改良中的利用研究[J].广西农业生物科学,2003,22(2)：75-83.
[19]YangHY, Ren X A, Weng QM, et al. Molecular mapping and genetic analysis of a rice brown planthopper resistance gene[J]. Hereditas,2002,136:39-43.
[20]陈大洲,邓仁根,肖叶青,等.东乡野生稻苗期耐寒性遗传研究.江西农业大学学报,1997,19(4)：56-59.
[21]CHEN Da-Zhou,XIAO Ye-Qing,ZHAO She-Xiang,PI Yong-Hua,XIONG Huan-Jin,LUO Li-Jun.Genetic study on the cold tolerance of dongxiang wild rice at the seedling stage.Acta Agri Univ Jiangxiensis(Nature Science),1997,19(4):56-59.
[22]庞汉华,陈成斌.中国野生稻资源[M].南宁：广西科学技术出版社,2002.
[23]HeHH, LiH B, Hu DL etal Varietal Improvement Utilizing Cold Tolerance Genes of Dongxiang Wild Rice [J]. Proceedings of InternationalRice Cold Resistance, Canberra Australia,2004,23-27.
[24]陈成斌,杨庆文,林竞鸿等.合浦县野生稻资源现状调查及保护对策[J].植物遗传资源学报,2005,6(4)：437-440
[25][4]李克敌,广西野生稻原生境保护点建设的进展问题和对策[J].植物遗传资源学报,2008,9(2)：230-233
[26]庞汉华,刘旭.栽培稻与野生稻后代花粉植株的诱导及其性状表现[J].作物学报,2000,26(1)：47-52.
[27]李子先,刘国平,陈忠友.中国东乡野生稻遗传因子转移的研究[J].遗传学报,1994,21(2)：133-146.
[28]邓启云,袁隆平,梁凤山,等.野生稻高产基因及其分子标记辅助育种研究[J].杂交水稻,2004,19(1)：6-10.
[29]AbeH Yamaguehi-ShinozakiK, UraoT, IWasakiT, HosokawaD, ShinozakiK (1997) Role of AiabidoPsis MYC and MYB homoloys in drought-and abseisic aeid-regulated gene expression. PlantCellg:1859-1868
[30]刘强,张贵友,陈受宜,(2000)植物转录因子的结构与调控作用科学通报,45(14)：1465-1474
[31]Boulikas T, (1994) Putative nuclear lacalization signals (NLS) in protein trans-cription factors. J Cell Biochen.55:32-58
[32]Dehesh K, Smith L QTepperman J M, et al.(1995) Twin autonomous bipartite nuclear localization signals direct nuclear import of GT-2.Plant J.8:25-36
[33]Varagona M J, Schmidt R J, Raikhel N V, (1992) Nuclear localization signals required for nuclear targeting of the maize regulatory protein Opaque-2. Plant Cell. 4:1213-1227
[34]Lyck R, Harmening U, Hohfeld etal. (1997) Intracellular distribution and identi-fication of the nuclear localization signals of two plant heat-stress transcription 51 factors. Planta,202:117-125
[35]Schwechheimer C, Bevan M. The regulation of transcription factor activity in plants. Trend in Plant Science,1998,3(10):278-283
[36]Stockinger E J, Gilmour S J, Thomashow M F. Arabidopsis Thaliana CBF1 en-codes an AP2 domain-containing transcription Activator that binds to the C-repeat/ DRE, a cis-actingDNA regulatory element that stimulates transcription in response to low temperature and water deficit[J]. Proc NatlAcad Sci USA,1997,94:1035-1040.
[37]Timmusk S, Wagner EGH. The plant growth-promoting rhizobacteria Paeniba-cillus polymyxa induces changes in Arabidopsis thaliana gene expression:a possible connection between biotic and abiotic stress responses [J].Mol Plant Microbe Interact, 1999,12:951-959.
[38]Medina J, Bargues M,Terol J, et al. The Arabidopsis CBF gene family is com-posed of three gene sencoding AP2 domain-containing proteins whose expression is regulated by low temperature but not by abscisic acid or dehydration[J]. Plant Physiol, 1999,119(2):463-470.
[39]Jaglo K R, Kleff S, Amundsen K L,et al.Components of the Arabidopsis C-repeat /Dehydration Responsive element binding factor cold-response pathway are con- served in Brassica napus and other plant species[J]. Plant Physiol,2001,127, 910-917.
[40]Yang Y,Wu J, Zhu K, etal. Identification and characterization of two chrysan-themum (Dendron-themax moriforlium) DREB genes, belonging to the AP2/EREBP family[J]. Mol Biol Rep,2007,6.
[41]Kim Y H, Yang K S, Ryu S H,et al.Molecular characterization of a cDNA enco-ding DRE-binding transcription factor from dehydration-treated fibrous roots of sweetpotato [J].Plant Physiol Biochem,2008,46(2):196-204.
[42]Dubouzet JG, Sakuma Y,Ito Y, etal. OsDREB genes in rice, Oryzasativa L., encode transcription activators that function in drought-,high-salt-and cold-responsive gene expression [J].Plant J,2003,73 33:751-763.
[43]Qin F, Sakuma Y, Li J, etal. Cloning and functional analysis of a novel DREB1/ CBF transcription factor involved in cold-responsive gene expression in Zea mays L[J]. Plant Cell Physiol,2004,45:1042-52.
[44]Pellegrineschi A, Reynolds M,Pacheco M, etal. Stress induced expression in wheat of the Arabidopsis thaliana DREB1A gene delays water stress symptoms under greenhouse conditions [J]. Genome,2004,47:493-500.
[45]Oh S J,Song S I,Kim Y S,et al.Arabidopsis CBF3/DREB1A and ABF3 in trans-genic rice increased tolerance to abiotic stress without stunting growth [J]. Plant Phy-siol,2005,138:341-351.
[46]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 Biophys Res Commun,2007,353:299-305.
[47]Ning Liu, Nai-Qin Zhong, Gui-Ling Wang. Cloning and functional characteri-zation of PpDBF1 gene encoding a DRE-binding transcription factor from Physcomi-trella patens[J].Planta,2007,226:827-838.
[48]Seki M, Narusaka M, Yamaguchi-Shinozaki K, etal. Arabidopsis encyclopedia using full-length cDNAs and its application[J]. Plant physiology and Biochemistry, 2001,39:211-220.
[49]Yamaguchi-Shinozaki K, Kasuga M, Liu Q, etal. Biological mechanisms of drought stress response [J]. JIRCAS Working Report,2002,105:1-8.
[50]Iwasaki T,Kiyosue T,Yamaguchi Shinozaki K,etal.The dehydration inducible rd17 (cor47) geneand its promoter region inArabidopsis thaliana[J]. Plant Physiol, 1997,115:1287-1292.
[52]徐莎,胡军,陈宇红,等DREB转录因子研究进展[J].农业生物技术学报,2008,16(4)：706-713.
[53]Mark D A, Kazuhiko Y, Masaru O T, etal. A novel mode of DNA recognition by ap-sheet revealed by the solution structure of the GCC-box binding domain in com-plex with DNA[J]. The EMBO Journal,1998,17(18):5484-5496.
[54]秦红霞,宋玉霞,刘敬梅.植物DREB转录因子的研究进展及应用[J].生物技术通讯,2006,17(5)：780-783.
[55]Bray E A, (1997)Plant responses to water deficit.Trends Plant Sci.,2:48-54
[56]Xiong L M, Schumaker K S, Zhu J K, (2002)Cell signaling during cold, drought, and salt stress. Plant Cell,14:165-183
[57]Grover A, Kapoor A, Lakshmi O S, etal. (2001) Understanding molecular alpha-bets of the plant abiotic stress responses. Current Science,80(2):206-215
[58]Pelle grineschi A, Ribaut J M, Trethowan R,et al. (2002) Progress in the genetic engineeering of wheat for waterlimited conditions. JIRCAS Working Report,55-60
[59]Ramanjulu S, Bartels D, (2002) Drought-and desiccation-induced modulation of gene expression in plants. Plant, Cell & Environment,25(2),141-151
[60]Wang W X, Vinocur B, Altman A, (2003) Plant responses to drought, salinity and extreme temperatures:towards genetic engineering for stress tolerance. Planta.218:1-14
[61]Xiong L M and Zhu J K, (2002) Molecular and genetic aspects of plant responses to osmotic stress. Plant, Cell and Environment,25,131-139
[62]Guo H, Ecker J R, (2004) The ethylene signaling pathway:new insights. Curr-opin Plant Biol.7 (1):40-49
[63]Chehab E W,Patharkar O R,Hegeman A D,et al.(2004) Autophosphorylation and subcellular localization dynamics of a salt and water deficitin duced calcium depend-ent protein kinase from Ice Plant. Plant Physiology,135:1430-1446
[64]Cheng N H, Pittman J K, Zhu J K, etal.(2004)The protein kinase SOS2 activates the Arabidopsis Hantiporter CAX1 to integrate calcium transport and salt tolerance. J.Biol. Chem.,279:2922-2926
[65]Gong D, Guo Y, Schumaker K S, etal.(2004) The SOS3 family of calcium sen-sors and SOS2 family of protein kinases in Arabidopsis. Plant Physiol.13:919-92649
[66]Sanchez-Barrena M J, Martinez-Ripoll M,Zhu J K, etal. (2004) SOS3(salt overly sensitive 3) from Arabidopsis thaliana:expression, purification, crystallization and preliminary X-ray analysis. Acts Crystallogr D 60:1272-1274
[67]Sanchez-Barrena M J, Matinez-Ripoll M, Zhu J K, etal. (2005)The structure of the Arabidopsis thaliana SOS3:molecular mechanism of sensing calcium for salt stress response. J Mol Biol.345:1253-1264
[68]Shinozaki K,Yamaguchi-Shinozaki K, (1997) Gene expression and signal trans-duction in water-stress response. Plant Physiolo gy,115:327-334
[69]Shinozaki K, (1998) Molecular response to water stress in Arabidopsis thali-ana. [J].Plant Res.111:345-351
[2]傅立国.中国植物红皮书(第一册)[M].北京：科学出版社,1992,314～316
[3]史冬燕,杨明挚等.元江普通野生稻叶片cDNA文库的构建及部分基因片段分析[J].HEREDITAS(Beijing)2008,30(6):776-780
[4]鄂志国,郑传举等.中国野生稻资源及其在水稻抗性育种上的应用[J].中国稻米,2008,6(4)：3-6
[5]MERRILLED.Oryza sativaL[J].Philip Journ Sci,1917,12(2):2.
[6]范树国,张再君,刘林,等.中国野生稻的种类、地理分布及其特征特性综述[J].武汉植物学研究,2000,18(5)：417-425.
[7]燕惠民,贺益娥,湖南野生稻资源调查及其保护措施[J].种子,2007,1(26)：88-90
[8]钟代彬,罗利军,应存山.野生稻在栽培稻育种中的应用[J].种子,1995,(1)：25～29.
[9]江川,王金英,李清华.野生稻的优异特性及其在水稻育种中的利用[J].福建稻麦科技,2003,21(3)：8～10.
[10]彭绍裘,魏子生,毛昌祥,等.云南省疣粒野生稻、药用野生稻和普通野生稻多抗性鉴定.植物病理学报,1982,12(4)：58-60.
[10]章琦.普通野生稻抗白叶枯病新基因被正式命名为Xa-23[J].作物杂志,2003,(1)：20-20.
[11]潘海军,王春连,赵开军.水稻抗白叶枯病基Xa-23的PCR分子标记定位及辅助选择[J].作物学报,2003,29(4)：501-507.
[12]姜文正,涂英文,丁忠华,等.东乡野生稻研究[J].作物品种资源,988,(3)：1-4.
[13]徐羡明,曾列先,林壁润,等.普通野生稻种质资源对纹枯病的抗性鉴定.植物病理学报,1992,22(4)：300
[14]徐羡明,林壁润,曾列先,等.普通野生稻种质资源对细菌性条斑病的抗性鉴定.植物保护,1991,17(6)：4-5.
[15]覃瑞,魏文辉,宁顺斌,等.利用水稻BAC克隆对Gm-2和Gm-6在药用野生稻中的FISH定位[J].中国农业科学,2001,34(1)：1-4
[16]Ishii T,Brar DS,Multani DS,Khush GS.Molecular tagging of genes for brown planthopper resistance and earliness introgressed from Oryza australiensis into cultivated rice,O.sativa.Genome,1994,37(2):217-221.
[17]李青,罗善昱,黄润清,等.广西药用野生稻抗褐稻虱研究简报.广西植保,1988,15(3)：165-166.
[18]李荣柏,秦学毅,韦素美等.普通野生稻稻褐飞虱抗性在水稻改良中的利用研究[J].广西农业生物科学,2003,22(2)：75-83.
[19]YangHY, Ren X A, Weng QM, et al. Molecular mapping and genetic analysis of a rice brown planthopper resistance gene[J]. Hereditas,2002,136:39-43.
[20]陈大洲,邓仁根,肖叶青,等.东乡野生稻苗期耐寒性遗传研究.江西农业大学学报,1997,19(4)：56-59.
[21]CHEN Da-Zhou,XIAO Ye-Qing,ZHAO She-Xiang,PI Yong-Hua,XIONG Huan-Jin,LUO Li-Jun.Genetic study on the cold tolerance of dongxiang wild rice at the seedling stage.Acta Agri Univ Jiangxiensis(Nature Science),1997,19(4):56-59.
[22]庞汉华,陈成斌.中国野生稻资源[M].南宁：广西科学技术出版社,2002.
[23]HeHH, LiH B, Hu DL etal Varietal Improvement Utilizing Cold Tolerance Genes of Dongxiang Wild Rice [J]. Proceedings of InternationalRice Cold Resistance, Canberra Australia,2004,23-27.
[24]陈成斌,杨庆文,林竞鸿等.合浦县野生稻资源现状调查及保护对策[J].植物遗传资源学报,2005,6(4)：437-440
[25][4]李克敌,广西野生稻原生境保护点建设的进展问题和对策[J].植物遗传资源学报,2008,9(2)：230-233
[26]庞汉华,刘旭.栽培稻与野生稻后代花粉植株的诱导及其性状表现[J].作物学报,2000,26(1)：47-52.
[27]李子先,刘国平,陈忠友.中国东乡野生稻遗传因子转移的研究[J].遗传学报,1994,21(2)：133-146.
[28]邓启云,袁隆平,梁凤山,等.野生稻高产基因及其分子标记辅助育种研究[J].杂交水稻,2004,19(1)：6-10.
[29]AbeH Yamaguehi-ShinozakiK, UraoT, IWasakiT, HosokawaD, ShinozakiK (1997) Role of AiabidoPsis MYC and MYB homoloys in drought-and abseisic aeid-regulated gene expression. PlantCellg:1859-1868
[30]刘强,张贵友,陈受宜,(2000)植物转录因子的结构与调控作用科学通报,45(14)：1465-1474
[31]Boulikas T, (1994) Putative nuclear lacalization signals (NLS) in protein trans-cription factors. J Cell Biochen.55:32-58
[32]Dehesh K, Smith L QTepperman J M, et al.(1995) Twin autonomous bipartite nuclear localization signals direct nuclear import of GT-2.Plant J.8:25-36
[33]Varagona M J, Schmidt R J, Raikhel N V, (1992) Nuclear localization signals required for nuclear targeting of the maize regulatory protein Opaque-2. Plant Cell. 4:1213-1227
[34]Lyck R, Harmening U, Hohfeld etal. (1997) Intracellular distribution and identi-fication of the nuclear localization signals of two plant heat-stress transcription 51 factors. Planta,202:117-125
[35]Schwechheimer C, Bevan M. The regulation of transcription factor activity in plants. Trend in Plant Science,1998,3(10):278-283
[36]Stockinger E J, Gilmour S J, Thomashow M F. Arabidopsis Thaliana CBF1 en-codes an AP2 domain-containing transcription Activator that binds to the C-repeat/ DRE, a cis-actingDNA regulatory element that stimulates transcription in response to low temperature and water deficit[J]. Proc NatlAcad Sci USA,1997,94:1035-1040.
[37]Timmusk S, Wagner EGH. The plant growth-promoting rhizobacteria Paeniba-cillus polymyxa induces changes in Arabidopsis thaliana gene expression:a possible connection between biotic and abiotic stress responses [J].Mol Plant Microbe Interact, 1999,12:951-959.
[38]Medina J, Bargues M,Terol J, et al. The Arabidopsis CBF gene family is com-posed of three gene sencoding AP2 domain-containing proteins whose expression is regulated by low temperature but not by abscisic acid or dehydration[J]. Plant Physiol, 1999,119(2):463-470.
[39]Jaglo K R, Kleff S, Amundsen K L,et al.Components of the Arabidopsis C-repeat /Dehydration Responsive element binding factor cold-response pathway are con- served in Brassica napus and other plant species[J]. Plant Physiol,2001,127, 910-917.
[40]Yang Y,Wu J, Zhu K, etal. Identification and characterization of two chrysan-themum (Dendron-themax moriforlium) DREB genes, belonging to the AP2/EREBP family[J]. Mol Biol Rep,2007,6.
[41]Kim Y H, Yang K S, Ryu S H,et al.Molecular characterization of a cDNA enco-ding DRE-binding transcription factor from dehydration-treated fibrous roots of sweetpotato [J].Plant Physiol Biochem,2008,46(2):196-204.
[42]Dubouzet JG, Sakuma Y,Ito Y, etal. OsDREB genes in rice, Oryzasativa L., encode transcription activators that function in drought-,high-salt-and cold-responsive gene expression [J].Plant J,2003,73 33:751-763.
[43]Qin F, Sakuma Y, Li J, etal. Cloning and functional analysis of a novel DREB1/ CBF transcription factor involved in cold-responsive gene expression in Zea mays L[J]. Plant Cell Physiol,2004,45:1042-52.
[44]Pellegrineschi A, Reynolds M,Pacheco M, etal. Stress induced expression in wheat of the Arabidopsis thaliana DREB1A gene delays water stress symptoms under greenhouse conditions [J]. Genome,2004,47:493-500.
[45]Oh S J,Song S I,Kim Y S,et al.Arabidopsis CBF3/DREB1A and ABF3 in trans-genic rice increased tolerance to abiotic stress without stunting growth [J]. Plant Phy-siol,2005,138:341-351.
[46]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 Biophys Res Commun,2007,353:299-305.
[47]Ning Liu, Nai-Qin Zhong, Gui-Ling Wang. Cloning and functional characteri-zation of PpDBF1 gene encoding a DRE-binding transcription factor from Physcomi-trella patens[J].Planta,2007,226:827-838.
[48]Seki M, Narusaka M, Yamaguchi-Shinozaki K, etal. Arabidopsis encyclopedia using full-length cDNAs and its application[J]. Plant physiology and Biochemistry, 2001,39:211-220.
[49]Yamaguchi-Shinozaki K, Kasuga M, Liu Q, etal. Biological mechanisms of drought stress response [J]. JIRCAS Working Report,2002,105:1-8.
[50]Iwasaki T,Kiyosue T,Yamaguchi Shinozaki K,etal.The dehydration inducible rd17 (cor47) geneand its promoter region inArabidopsis thaliana[J]. Plant Physiol, 1997,115:1287-1292.
[52]徐莎,胡军,陈宇红,等DREB转录因子研究进展[J].农业生物技术学报,2008,16(4)：706-713.
[53]Mark D A, Kazuhiko Y, Masaru O T, etal. A novel mode of DNA recognition by ap-sheet revealed by the solution structure of the GCC-box binding domain in com-plex with DNA[J]. The EMBO Journal,1998,17(18):5484-5496.
[54]秦红霞,宋玉霞,刘敬梅.植物DREB转录因子的研究进展及应用[J].生物技术通讯,2006,17(5)：780-783.
[55]Bray E A, (1997)Plant responses to water deficit.Trends Plant Sci.,2:48-54
[56]Xiong L M, Schumaker K S, Zhu J K, (2002)Cell signaling during cold, drought, and salt stress. Plant Cell,14:165-183
[57]Grover A, Kapoor A, Lakshmi O S, etal. (2001) Understanding molecular alpha-bets of the plant abiotic stress responses. Current Science,80(2):206-215
[58]Pelle grineschi A, Ribaut J M, Trethowan R,et al. (2002) Progress in the genetic engineeering of wheat for waterlimited conditions. JIRCAS Working Report,55-60
[59]Ramanjulu S, Bartels D, (2002) Drought-and desiccation-induced modulation of gene expression in plants. Plant, Cell & Environment,25(2),141-151
[60]Wang W X, Vinocur B, Altman A, (2003) Plant responses to drought, salinity and extreme temperatures:towards genetic engineering for stress tolerance. Planta.218:1-14
[61]Xiong L M and Zhu J K, (2002) Molecular and genetic aspects of plant responses to osmotic stress. Plant, Cell and Environment,25,131-139
[62]Guo H, Ecker J R, (2004) The ethylene signaling pathway:new insights. Curr-opin Plant Biol.7 (1):40-49
[63]Chehab E W,Patharkar O R,Hegeman A D,et al.(2004) Autophosphorylation and subcellular localization dynamics of a salt and water deficitin duced calcium depend-ent protein kinase from Ice Plant. Plant Physiology,135:1430-1446
[64]Cheng N H, Pittman J K, Zhu J K, etal.(2004)The protein kinase SOS2 activates the Arabidopsis Hantiporter CAX1 to integrate calcium transport and salt tolerance. J.Biol. Chem.,279:2922-2926
[65]Gong D, Guo Y, Schumaker K S, etal.(2004) The SOS3 family of calcium sen-sors and SOS2 family of protein kinases in Arabidopsis. Plant Physiol.13:919-92649
[66]Sanchez-Barrena M J, Martinez-Ripoll M,Zhu J K, etal. (2004) SOS3(salt overly sensitive 3) from Arabidopsis thaliana:expression, purification, crystallization and preliminary X-ray analysis. Acts Crystallogr D 60:1272-1274
[67]Sanchez-Barrena M J, Matinez-Ripoll M, Zhu J K, etal. (2005)The structure of the Arabidopsis thaliana SOS3:molecular mechanism of sensing calcium for salt stress response. J Mol Biol.345:1253-1264
[68]Shinozaki K,Yamaguchi-Shinozaki K, (1997) Gene expression and signal trans-duction in water-stress response. Plant Physiolo gy,115:327-334
[69]Shinozaki K, (1998) Molecular response to water stress in Arabidopsis thali-ana. [J].Plant Res.111:345-351