冯虎;张颖;樊秀彩;姜建福;孙海生;刘崇怀;

• 1:中国农业科学院郑州果树研究所

摘要(Abstract)：

【目的】获得来源于中国野生抗病种质刺葡萄0943的转录因子Vd WRKY49基因,揭示其序列特征、基因功能与抗葡萄白腐病之间的关系,初步揭示抗病种质抗病机制。【方法】利用同源克隆的方法分别获得刺葡萄0943的转录因子Vd WRKY49基因的编码区和启动子区域,分析其序列特征,通过实时荧光定量检测其对白腐病菌和水杨酸诱导的反应,同时以感病品种欧亚种‘黑比诺’为对照,分析不同种质中的转录因子Vd WRKY49基因序列及表达差异。【结果】来源于中国野生种质刺葡萄0943的Vd WRKY49基因,在其编码区和启动子区域都有抗病基因的序列特征和位点特征,同时也存在不同,在DNA和氨基酸水平上与来源于欧亚种的‘黑比诺’Vv WRKY49有差异,这些差异可能造成了其结构功能的差异;受到葡萄白腐病和水杨酸诱导后,Vd WRKY49基因的表达量明显高于Vv WRKY49。【结论】来源于刺葡萄0943的Vd WRKY49基因受到白腐病菌和水杨酸诱导后表达量和表达模式明显区别来源于‘黑比诺’的Vv WRKY49基因,推测在葡萄抗病途径中转录因子WRKY49具有重要的生物学作用。

关键词(KeyWords)： 中国野生刺葡萄0943;转录因子;WRKY49;白腐菌

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金(31201599);; 国家葡萄产业技术体系(CARS-30);; 中国农业科学院科技创新工程专项经费项目(CAASASTIP-2015-ZFRI)

作者(Authors): 冯虎;张颖;樊秀彩;姜建福;孙海生;刘崇怀;

DOI: 10.13925/j.cnki.gsxb.20150050

参考文献(References)：

• [1]LIU Chonghuai,KONG Qingshan.Preservation condition of Chi?na wild grape resources and advice[J].Sino-Overseas Grapevine&Wine,2008(1):42-46.刘崇怀,孔庆山.我国野生葡萄资源的保存现状及建议[J].中外葡萄与葡萄酒,2008(1):42-46.
• [2]HE Puchao,WANG Yuejin,WANG Guoying,REN Zhibang,HE Chuncheng.The studies on the disease-resistance of Vitis wild species originated in China[J].China Academic Journal,1991,24(3):50-56.贺普超,王跃进,王国英,任治邦,和纯成.中国葡萄属野生种抗病性的研究[J].中国农业科学,1991,24(3):50-56.
• [3]ZHANG Ying,SUN Haisheng,FAN Xiucai,JIANG Jianfu,LIU Chonghuai.Identification and evaluation of resistance of Vitis to grape white rot[J].Journal of Fruit Science,2013,30(2):191-196.张颖,孙海生,樊秀彩,姜建福,刘崇怀.中国野生葡萄资源抗白腐病鉴定及抗性种质筛选[J].果树学报,2013,30(2):191-196.
• [4]EULGEM T,RUSHTON P J,ROBATZEK S,SOMSSICH I E.The WRKY superfamily of plant transcription factors[J].Trends in Plant Science,2000,5(5):199-206.
• [5]JIANG Y,DEYHOLOS M K.Functional characterization of arabi?dopsis Na Cl-inducible WRKY25 and WRKY33 transcription fac?tors in abiotic stresses[J].Plant Molecular Biology,2009,69(1-12):91-105.
• [6]TAO Z,KOU Y,LIU H,LI X,XIAO J,WANG S.Os WRKY45alleles play different roles in abscisic acid signalling and salt stress tolerance but similar roles in drought and cold tolerance in rice[J].Journal of Experimental Botany,2011,62(14):4863-4874.
• [7]LI Huie,GUO Qiqiang,LAN Xiaozhong,ZHOU Qi,WEI Na.Comparative expression analysis of five WRKY genes from Tibet?an Hulless Barley under various abiotic stresses between droughtresistant and sensitive genotype[J].Acta Physiologiae Plantarum,2014,36(4):963-973.
• [8]DELLAGI A,HELIBRONN J,AVROVA A O,MONTESANO M,PALVA E T,STEWART H E,TOTH I K,COOKE D E,LY?ON G D,BIRCH P R.A potato gene encoding a WRKY-like transcription factor is induced in interactions with Erwinia caroto?vora Subsp.atroseptica and phytophthora infestans and is coregu?lated with Class I Endochitinase expression[J].Mol Plant Microbe Interact,2000,13(10):1092-1101.
• [9]DESLANDES L,OLIVIER J,THEULIERES F,HIRSCH J,FENG D X,BITTNER-EDDY P,BEYNON J,MARCO Y.Re?sistance to ralstonia solanacearum in Arabidopsis thaliana is Con?ferred by the recessive RRS1-R gene,a member of a novel fami?ly of resistance genes[J].Proceedings of the National Academy of Sciences USA,2002,99(4):2404-2409.
• [10]LAN A,HUANG J,ZHAO W,PENG Y,CHEN Z,KANG D.A salicylic acid-induced rice(Oryza sativa L.)transcription factor Os WRKY77 is involved in disease resistance of Arabidopsis thali?ana[J].Plant Biology,2013,15(3):452-461.
• [11]LEE H,KO Y J,CHA J Y,PARK S R,AHN I,HWANG D J.The C-terminal region of Os WRKY30 is sufficient to confer en?hanced resistance to pathogen and activate the expression of de?fense-related genes[J].Plant Biotechnology Reports,2013,7(3):221-230.
• [12]PAN L J,JIANG L.Identification and expression of the WRKY transcription factors of Carica papaya in response to Abiotic and Biotic stresses[J].Molecular Biology Reporter,2014,41(3):1215-1225.
• [13]ZHANG C,GROSIC S,WHITHAM S A,HILL J H.The Re?quirement of multiple defense genes in Soybean Rsv1-mediated extreme resistance to Soybean Mosaic Virus[J].Mol Plant Microbe Interact,2012,25(10):1307-1313.
• [14]LIM J H,PARK C J,HUH S U,CHOI L M,LEE G J,KIM Y J,PAEK K H.Capsicum annuum WRKYb transcription factor that binds to the Ca PR-10 promoter functions as a positive regula?tor in innate immunity upon TMV infection[J].Biochemical and Biophysical Research Communications,2011,411(3):613-619.
• [15]ZHANG Ying,FENG Jiancan.Identification and characterization of the grape WRKY family[J].Bio Med Research International,2014,2014:787680.
• [16]MARCHIVE C,MZID R,DELUC L,BARRIEU F,PIRRELLO J,GAUTHIER A,CORIO-COSTET M F,REGAD F,CAILLE?TEAU B,HAMDI S,LAUVERGEAT V.Isolation and character?ization of a Vitis vinifera transcription factor,Vv WRKY1,and its effect on responses to fungal pathogens in transgenic tobacco plants[J].Journal of Experimental Botany,2007,58(8):1999-2010.
• [17]MZID R,MARCHIVE C,BLANCARD D,DELUC L,BAR?RIEU F,CORIO-COSTET M F,DRIRA N,HAMDI S,LAU?VERGEAT V.Overexpression of Vv WRKY2 in tobacco enhances broad resistance to necrotrophic fungal pathogens[J].Physiologia Plantarum,2007,131(3):434-447.
• [18]LI Huie,XU Yan,XIAO Yu,ZHU Ziguo,XIE Xiaoqing,ZHAO Heqing,WANG Yuejin.Expression and functional analy?sis of two genes encoding transcription factors,Vp WRKY1 and Vp WRKY2,isolated from Chinese Wild Vitis Pseudoreticulata[J].Planta,2010,232(6):1325-1337.
• [19]ZHU Ziguo,SHI Jiangli,CAO Jiangling,HE Mingyang,WANG Yuejin.Vp WRKY3,a Biotic and Abiotic stress-related transcrip?tion factor from the Chinese Wild Vitis Pseudoreticulata[J].Plant Cell Reports,2012,31(11):2109-2120.
• [20]YU Yihe,XU Weirong,WANG Jie,WANG Lei,YAO Wenkong,YANG Yazhou,XU Yan,MA Fuli,DU Yangjian,WANG Yuejin.The Chinese wild grapevine(Vitis pseudoreticulata)E3 ubiquitin ligase Erysiphe necator-induced RING finger protein 1(EIRP1)activates plant defense responses by inducing proteolysis of the Vp WRKY11 transcription factor[J].New Phytologist,2013,200(3):834-846.
• [21]MANDAL S,MALICK N,MITRA A.Salicylic acid-Induced re?sistance to Fusarium oxysporum f.sp.lycopersici in tomato[J].Plant Physiol Biochem,2009,47(7):642-649.
• [22]WALKER J M,GASTEIGER E,HOOGLAND C,GATTIKER A,DUVAUND S,WILKINS M R,APPEL R D,BAIROCH A.Protein identification and analysis tools on the Ex PASy Server[M].USA:Humana Press,2005:571-607.
• [23]KELLEY L A,STERBERG M J E.Protein structure prediction on the Web:a case study using the Phyre Server[J].Nature Proto?cols,2009,4(3):363-371.
• [24]MARFORI M,MYNOTT A,ELLIS J J,MEHDI A M,SAUN?DERS N F W,CURMI P M,FORWOOD J K,BODEN M,KO?BE B.Molecular basis for specificity of nuclear import and predic?tion of nuclear localization[J].Biochimica et Biophysica Acta(BBA)-Molecular Cell Research,2011,1813(9):1562-1577.
• [25]REESE M G.Application of a time-delay neural network to pro?moter annotation in the drosophila melanogaster genome[J].Com?puters&Chemistry,2001,26(1):51-56.
• [26]HIGO K,UGAWA Y,IWAMOTO M,KORENAGA T.Plant cisacting regulatory DNA elements(PLACE)database:1999[J].Nu?cleic Acids Res,1999,27(1):297-300.
• [27]PRESTRIDGE D S.SIGNAL SCAN:A computer program that scans DNA sequences for eukaryotic transcriptional elements[J].Comput Appl Biosci,1991,7(2):203-206.
• [28]KIM C Y,ZHANG S Q.Activation of a Mitogen-Activated pro?tein kinase Cascade induces WRKY family of transcription fac?tors and defense genes in tobacco[J].The Plant Journal,2004,38(1):142-151.
• [29]LIU Y L,SCHIFF M,DINESH-KUMAR S P.Involvement of MEK1 MAPKK,NTF6 MAPK,WRKY/MYB transcription factors[J].The Plant Journal,2004,38(5):800-809.
• [30]MIAO Y,LAUN T M,SMYKOWSKI A,ZENTGRAF U.Arabi?dopsis MEKK1 can take a short cut:it can directly interact with senescence-related wrky53 transcription factor on the protein lev?el and can bind to its promoter[J].Plant Molecular Biology,2007,65(1-2):63-76.
• [31]CHISHOLM S T,COAKER G,DAY B,STASKAWICZ B J.Host-microbe interactions:shaping the evolution of the plant im?mune response[J].Cell,2006,124(4):803-814.
• [32]EULGEM T,IMRE E.SOMSSICH.Networks of WRKY transcrip?tion factors in defense signaling[J].Current Opinion in Plant Biol?ogy,2007,10(4):366-371.
• [33]üLKER B,SOMSSICH I E.WRKY transcription factors:from DNA binding towards biological function[J].Current Opinion in Plant Biology,2004,7(5):491-498.
• [34]CAI Meng,QIU Deyun,YUAN Ting,DING Xinhua,LI Hon?gjing,DUAN Liu,XU Caiguo,LI Xianghua,WANG Shiping.Identification of novel pathogen-responsive cis-elements and their binding proteins in the promoter of Os WRKY13,a gene reg?ulating rice disease resistance[J].Plant,Cell&Environment,2008,31(1):86-96.
• [35]ISHIHAMA N,YOSHIOKA H.Post-translational regulation of WRKY transcription factors in plant immunity[J].Current Opin?ion in Plant Biology,2012,15(4):431-437.
• [36]WHITESIDE S T,GOODBOURN S.Signal transduction and nu?clear targeting:regulation of transcription factor activity by Sub?cellular Localisation[J].Journal of Cell Science,1993,104(Pt 4):949-955.
• [37]HAAKE V,COOK D,RIECHMANN J L,PINEDA O,THOM?ASHOW M F,JAMES Z.Transcription factor CBF4 is a regulator of drought adaptation in Arabidopsis[J].Plant Physiology,2002,130(2):639-648.
• [38]DIPAK P R,PELAGIA F.CCAAT/enhancer-binding proteins:structure,function and regulation[J].Biochemical Journal,2002,365:561-575.
• [39]SAKURMA Y,LIU Q,DUBOUZET J G,ABE H,SHINOZAKI K,YAMAGUCHI-SHINOZAKI K.DNA-Binding specificity of the ERF/AP2 Domain of Arabidopsis DREBs,transcription fac?tors involved in dehydration-and cold-inducible gene expression[J].Biochemical and Biophysical Research Communications,2002,290(3):998-1009.
• [40]OHSHIMA M,ITOH H,MATSUOKA M,MURAKAMI T,OHASHI Y.Analysis of stress-induced or salicylic acid-induced expression of the pathogenesis-related 1a protein gene in trans?genic tobacco[J].The Plant Cell Online,1990,2(2):95-106.
• [41]RUSHTON P J,TORRES J T,PARNISKE M,WERNERT P,HAHLBROCK K,SOMSSICH I E.Interaction of elicitor-in?duced DNA-binding proteins with elicitor response elements in the promoters of parsley PR1 genes[J].The EMBO Journal,1996,15(20):5690.
• [42]EULGEM T,RUSHTON P J,SCHMELZR E,HAHLBROCK K,SOMSSICH I E.Early nuclear events in plant defence signalling:rapid gene activation by WRKY transcription factors[J].The EM?BO Journal,1999,18(17):4689-4699.
• [43]YANG Peizhen,CHEN Chunhong,WANG Zeping,FAN Bao?fang,CHEN Zhixiang.A pathogen and salicylic acid induced WRKY DNA binding activity recognizes the elicitor response ele?ment of the tobacco class I chitinase gene promoter[J].The Plant Journal,1999,18(2):141-149.
• [44]CHEN Chunhong,CHEN Zhixiang.Isolation and characteriza?tion of two pathogen-and salicylic acid-induced genes encoding WRKY DNA-binding proteins from tobacco[J].Plant Molecular Biology,2000,42(2):387-396.