摘要
植物的生长和发育常常会受到逆境胁迫的影响,逆境胁迫包括生物胁迫与非生物胁迫。这些逆境胁迫对植物的生长发育及作物的与品质造成了严重的影响。而在胁迫发生时,植物感知并传递胁迫信号,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,使植物在一定程度上能够抵御胁迫对其的伤害。大量研究表明,植物AP2/EREBP类转录因子对于植物应答外界胁迫及提高植物的抗逆性方面起着极为重要的作用。因此研究ERF类转录因子的作用机制具有重要的理论与实践意义。
采用同源克隆法从构建的非亲和小种侵染诱导的小麦cDNA文库(由中国农业科学院植物保护研究所麦类作物病害课题组构建)中得到了两个小麦ERF类转录因子基因的全长cDNA克隆,命名为TaERF4、TaERF5。根据其预测的氨基酸序列分析表明,它们含有一个保守的AP2结构域,与已公布的ERF亚家族成员有较高同源性。同源性分析表明,TaERF4、TaERF5与典型的ERF亚家族成员在进化树上的距离较近,TaERF4/5可能是一个新的AP2/EREBP转录因子基因。半定量RT-PCR分析结果表明,TaERF5对脱落酸ABA做出了应答、TaERF4、TaERF5对茉莉酸甲酯MeJA做出了应答,在经处理后,其转录水平升高；在低温、干旱和高盐处理中,两个基因的表达水平均有显著的提高。利用基因枪转化的方法对TaERF4/5的亚细胞定位进行了研究,结果表明TaERF4/5具有核定位功能。
通过以上实验表明,TaERF4/5因具有AP2/EREBP类转录因子基因的基本特征。TaERF4/5基因受到低温、干旱及高盐等非生物胁迫的诱导。这些研究结果为小麦的分子抗性育种提供了理论基础,具有重要的实际意义。
Plant growth and development are invariably influenced by various abiotic and biotic stresses, and those environmental stresses seriously limit the increase of yield and improvement of quality of crops. However, plants could sense and transduce the signals caused by stress to transcription factors through a series of phosphorization responses. The functional genes related to stress tolerance are then initiated; a series of physiochemical reactions are activated to alleviate the damage caused by abiotic stress to a certain degree. Extensive research suggests that plant AP2/EREBP superfamily transcription factors played important roles in response to environment stresses. Therefore, to study the ERF transcription factors contain important significance in understand the mechanism of plant response to environment stresses.
A full lengh cDNA designated as TaERF4, TaERF5 was isolated from a wheat cDNA library (the State Key Laboratory for Biology of Plant Diseases and Pest Insects, Institute of plant Protection, Chinese Academy of Agricultural Sciences) using the near-isogenetic line Taichung29*6/Yr5 seedlings challenged by incompatible yellow rust race CY32 through homology-based cloning method. The amino acid sequenced contains of TaERF4/5 contains a highly conserved AP2 domain, which shares quite high homology with members of ERF subfamily. The cluster analysis showed that TaERFL4/5 belong to ERF subfamily, thus TaERF4/5 are probably a new member of ERF subfamily genes. Semi-quantitative RT-PCR analysis revealed that the expression of TaERF5 was induced by ABA (abscisic acid), TaERF4, TaERF5 were induced by MeJa (methyl jasmonate), cold, salt and dehydration respectively. We studied the subcellular localization of TaERF4/5 using PDS 1000/He Gene System(Bio-Rad), results show that TaERF4/5 can localize into the nuclei. These results suggest that TaERF4/5 was AP2/EREBP transcription factor with basic characteristics, and it may be involved in process of plant response to both abiotic and biotic stresses. The experiment offered theoretical foundation to molecular breeding of wheat.
引文
[1]Lyons J M and Raison J K. Oxidative activity of mitochondria isolated from plant tissues sensitive and resistant to chilling injury[J]. Plant Physiol,1970,45:386-389.
[2]陈辉蓉,吴振斌,贺锋等.植物抗逆性研究进展[J].环境污染治理技术与设备,2001,2(3)：7-13.
[3]Levitt J. Responses of Plants to Environmental Stresses [M]. Volume Ⅱ 2nd. New York:Academic Press,1980.
[4]刘强,赵南明,Yamaguch—Shinozaki K.等.DREB转录音因子在提高植物抗逆性中的作用[J].科学通报,2000,45(1)：11-16.
[6]Singh K B. Transcriptional regulation in plant:The importance of combinatorial control. Plant Physiol,1998,18:1111-1120.
[7]Meshi T, wabuchi M. Plant transcription factors. Plant Cell physiol,1995,36 (8):1405-1420.
[8]Liu L, White M J, MacRae T H. Transcription factors and their genes in higher plants. Eur J Biochem,1999,262:247-257.
[9]Sakuma Y, Liu Q, Dubouzet J G et al. DNA-Binding specificity of the ERF AP2 domain of Arabidopsis DREBs, transcription factors involved in dehydration and cold inducible gene expression[J]. Plant biology,2002,998-1009.
[10]Jofuku K D, Boer B G W, Montagu M V et al. Control of Arabidopsis flower and seed development by the homeotic gene APETALA2[J]. Plant biology,1994, 1211-1225.
[11]Jofuku K D, Omidyar P K, Gee Z et al. Control of seed yield by the floralhomeotic gene APETALA2[J]. Pro natl Acad Sci,2005,3117-3122.
[12]Elliott R C, Betzner A S, Huttner E et al. AINTEGUMENTA, an APETALA2-like gene of Arabidopsis with pleiotropic role in ovule development and floral organ growth. Plant biology,1996,155-168.
[13]Liu Q, Kasuga M, Sakuma Y et al. Two transcription factors, DREB1 and DREB, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought and low temperature responsive gene expression, respectively, in Arabidopsis[J]. Plant biology,1998,1391-1406.
[14]Kagaya Y, Ohmiya K, Hattori T.RAV1, a novel DNAbinding protein, binds to bipartite recognition sequence through two distinct DNA-binding domains uniquely found in higher plants[J]. Nucleic Acids Res,1999,27:470-478.
[15]Chinnusamy V, Ohta M, Kanrar S et al. ICE1:a regulator of cold-induced transcriptome and freezing tolerance in Arabidopsis[J]. Genes & Development, 2003,17:1043-1054.
[16]Fujimoto S Y, Ohta M, Usui A et al. Arabidopsis ethylene-responsive element binding factors act as transcriptional activator or repressors of GCC box-mediated gene expression. Plant biology,2000,393-404.
[17]Brown R L, Kanzan K, MaGrath K C et al. A role for the GCC-box in jasmonate-mediated activation of the PDF1.2 gene of Arabidopsis[J]. Plant Biol,2003, 1020-1032.
[18]Chen C H, Chen Z X. Potentiation of developmentally regulated plant defense response by AtWRKY18, a pathogen induced Arabidopsis transcription factor [J]. Plant Physiol,2002,706-716.
[19]Gutterson N and Reuber T L. Regulation of disease resistance pathways by AP2/ERF transcription factors[J]. Plant biology,2004,465-471.
[20]Onate-Sanchez L and Singh K B. Identification of Arabidopsis ethylene-responsive element binding factors with distinct induction kinetics after pathogen infection[J]. Plant biology,2002,1313-1322.
[21]Tournier B, Sanchez-Ballesta M, Jones B et al.New members of the tomato ERF family show specific expression pattern and diverse DNA binding capacity to the GCC box element[J]. Plant biology,2003,149-154.
[22]Aharoni A, Dixit S, Jetter R et al. The SHINE clade of AP2 domain transcription factors activates wax biosynthesis, alters cuticle properties, and confers drought tolerance when overexpresson in Arabidopsis[J]. Plant Cell,2004,16, 2463-2480.
[23]Zhang J Y, Broeckling C D, Blancaflor E B.Overexpression of WXP1, a putative Medicago truncatula AP2 domain-containing transcription factor gene, increases cuticular wax accumulation and enhances drought tolerance in transgenic alfalfa(Medicago sativa)[J]. Plant biology,2005,42:689-707.
[24]Ohta M, Matsui K, Hiratsu K et al. Repression domains of class Ⅱ ERF transcriptional repressors share an essential motif for active repression[J]. Plant Cell,2001,13:1959—1968.
[25]Zhou x, Riehon V M, Rifkind R A et al. Identification of a transcriptional repressor related to the noneatalytie domain of histone deacetylases 4 and 5 [J]. Proe. Natl. Acad. Sci. USA,2000,97(3)1056—1061.
[26]Eyal Y, Meller Y, Le v-Yadun S et al. A basic-type PR-1 promoter directs ethylene responsiveness, vascular and abscission zone-specific expression [J]. Plant J, 1993,4:225-234.
[27]Ohme-TakagiM, ShinshiH. Ethylene-inducible DNA binding proteins that interact with an ethylene-responsive element[J].Plant Cell,1995,7:173-182.
[28]Pirrello J, Jaimes-Miranda F, Sanchez-Ballesta M T et al. S1-ERF2, a tomato ethylene response factor involved in ethylene response and seed germination[J], Plant Cell Physiol,2006,47:1195-1205.
[29]Gu YQ, Wildermuth M C, Chakravarthy S et al. Tomato transcription factors pti4, pti5, and pti6 activate defense responses when expressed in Arabidopsis[J]. Plant Cell 14,2002,817-831.
[30]Ham B K, Park J M, Lee S B et al. Tobacco Tsipl, a DnaJ-type Zn finger protein, is recruited to and potentiates Tsil-mediated transcriptional activation[J]. Plant Cell,2006,18,2005-2020.
[31]Tang M, Sun J, Liu Y et al. Isolation and functional characterization of the JcERF gene, a putative AP2/EREBPdomain-containing transcription factor, in the woody oil plant Jatropha curcas[J]. Plant Mol Biol,2006,63,419-428.
[32]Onate-Sanchez L, Anderson J P, Young J et al. AtERF14, a member of the ERF family of transcription factors, plays a non-redundant role in plant defense [J]. Plant Physiol,2007,143,400-409.
[33]Sohn K H, Lee S C, Jung H W et al. Expression and functional roles of the pepper pathogen-induced transcription factor RAVI in bacterial disease resistance, and drought and salt stress tolerance[J]. Plant Mol Biol,2006,61,897-915.
[34]舒群芳.植物基因克隆的方法和策略[J].植物学通报,1996,16(1)：80-85.
[35]易图永,谢丙炎,张宝玺等.植物抗病基因同源序列及其在抗病基因克隆与定位中的应用[J].生物技术通报,2002,(2)：16-20.
[36]Catherine F, Cabriele S, Beat k. et al. Molecular cloning of a new receptor-like kinase gene encoded at the Lr10 disease resistance locus of wheat [J]. The plant Journal,1997,11(1):45-52.
[37]Katherine A S, Blake C K, Nurul M I F et al. Resistance gene candidates identified by PCR with degenerate oligonucleotide primers map to dusters of resistance gene in lettuce[J]. MIPI,1998,11(8):815-823.
[38]Leister D, Ballvora A, Saamini F et al. A PCR-based approach for isolating pathogen resistance genes from potato with potential for wicle application in plants[J]. Nat Genet,1996,14:421-429.
[39]Yong G Y, Glenn R B, Saghaim M A.1996. Isolation of a superfamily of candidate disease-resistance genes in soybean based on a conserved nucleotide-binding site[J]. Proe Natl Acad Sci USA,1996,93:11751-11756.
[40]Kanazin V, Marek L F. Shoemaker R. Resistance gene analogues are conserved and clustered in soybean[J]. Proe Natl Acad Sci USA,1996,93:11746-11750.
[41]Leister D, Kurth J, Laurie D A et al. Rapid reorganization of resistance gene homologues in cereal genomes[J]. Proe Natl Acad Sci USA,1998,95:370-375.
[42]Li XQ, Liu CN, Ritchie SW, et al. Factors influencing Agrobacterium-mediated transient expression of gusA in rice[J]. Plant Mol Biol 1992;20(6):1037-48.
[43]Hara-Nishimura I, Matsushima R. A wound-inducible organelle derived from endoplasmic reticulum:a plant strategy against environmental stresses [J] Curr Opin Plant Biol 2003;6 (6):583-8.
[44]Mizuno S, Hirasawa Y, Sonoda M, et al. Isolation and characterization of three DREB/ERF-type transcription factor from melon (Cucunis melo). Plant Science, 2006,170:1156-1163.