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[1]孟凡姗,范素杰,徐鹏飞,等.大豆GmPＲ10 基因启动子的克隆及序列分析[J].大豆科学,2016,35(04):568-573.[doi:10.11861/j.issn.1000-9841.2016.04.0568]
　MENG Fan-shan,FAN Su-jie,XU Peng-fei,et al.Cloning and Sequence Analysis of the GmPＲ10 Gene Promoter from Soybean( Glycine max L. )[J].Soybean Science,2016,35(04):568-573.[doi:10.11861/j.issn.1000-9841.2016.04.0568]

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大豆GmPＲ10 基因启动子的克隆及序列分析

《大豆科学》[ISSN:1000-9841/CN:23-1227/S] 卷: 第35卷期数: 2016年04期页码: 568-573 栏目: 出版日期: 2016-07-20

Title:: Cloning and Sequence Analysis of the GmPＲ10 Gene Promoter from Soybean( Glycine max L. )

作者:: 孟凡姗; 范素杰; 徐鹏飞; 张淑珍; ( 东北农业大学大豆研究所/大豆生物学教育部重点实验室，黑龙江哈尔滨150030)

Author(s):: MENG Fan-shan; FAN Su-jie; XU Peng-fei; ZHANG Shu-zhen; ( Soybean Ｒesearch Institute of Northeast Agricultural University/Key Laboratory of Soybean Biology of Chinese Ministry of Education，Harbin 150030，China)

关键词:: 大豆; GmPＲ10; 启动子; 克隆; GUS 染色

Keywords:: Soybean; GmPＲ10; Promoter; Cloning; GUS staining

分类号:: S565. 1

DOI:: 10.11861/j.issn.1000-9841.2016.04.0568

文献标志码:: A

摘要:: GmPＲ10 基因是病程相关蛋白PＲ10( pathogenesis-related proteins 10) 在大豆中的同源基因。为探明大豆Gm-PＲ10 基因的表达调控规律，应用PCＲ技术从大豆抗疫霉根腐病品种绥农10 号中克隆了GmPＲ10 基因上游2 235 bp的启动子序列pGmPＲ10，定向替换pBI121 载体的CaMV35S 组成型启动子，构建植物表达载体pBI121 /pGmPＲ10 /GUS，并转化农杆菌侵染烟草叶盘。GUS 染色结果表明，pGmPＲ10 受聚乙二醇( PEG) 、低温( 4℃) 、水杨酸( SA) 、茉莉酸( JA) 和脱落酸( ABA) 诱导表达，因此推测GmPＲ10 基因可能参与植物激素调节植物生长发育的过程，以及生物胁迫和非生物胁迫条件下植物对环境响应的过程。此外，利用PLACE 和PlantCAＲE 在线启动子预测工具分析pGm-PＲ10，结果表明: pGmPＲ10 含有启动子的一般结构TATA-box 和CAAT-box，光应答元件，生长素和细胞分裂素响应元件，热激元件，低温应答元件，干旱应答元件以及ABA、SA、JA 应答元件等。

Abstract:: GmPＲ10 was homology with the PＲ10 ( pathogenesis-related proteins 10) gene in other plants. To investigate theexpression and regulation of the GmPＲ10 gene in soybean，the 2 235 bp promoter region of 5’-flanking upstream of GmPＲ10gene was isolated from the genomic DNA of soybean cultivars Suinong 10( with high resistance to Phytophthora sojae in Heilongjiang，China) by PCＲ，defined as pGmPＲ10. It was directionally replaced CaMV35S promoter of the expression vectorpBI121. The plant expression vector of pBI121 /pGmPＲ10 /GUS was used to drive expression of the GUS reporter gene in thepBI121，and the construct was transformed into tobacco leaves by Agrobacterium-mediated transformation. GUS staining resultsshowed that，pGmPＲ10 activity was highly induced by drought ( PEG) ，low temperature ( 4℃) ，salicylic acid ( SA) ，jasmonicacid ( JA) and abscisic acid ( ABA) . Therefore，it was speculated that GmPＲ10 gene may be involved in the process ofplant growth and development with the regulation of plant hormones，as well as the process under abiotic and abiotic stressesresponses to the environment. In addition，sequence analysis by PLACE and PlantCAＲE revealed that pGmPＲ10 containedpromoter general structure TATA-box and CAAT-box，light response element，auxin and cytokinin response element，heatshock element，cold response element，drought responsive element and ABA，SA and JA response elements.

参考文献/References:

［1］ van Loon L C，Van Strien E A. The families of pathogenesis-relatedproteins，their activities，and comparative analysis of PＲ-1 typeproteins［J］. Physiological and Molecular Plant Pathology，1999，55: 85-97.

［2］ van Loon L C，Ｒep M，Pieterse C M J. Significance of inducibledefense-related proteins in infected plants［J］. Annual Ｒeview ofPhytopathology，2006，44: 135-162.

［3］ Czermic P，Huang H C，Marco Y. Characterization of hsr201 andhsr515，two tobacco genes preferentially expressed during the hypersensitivereaction provoked by phytopathogenic bacteria［J］.Plant Molecular Biology，1996，31( 2) : 255-265.

［4］ Lim S，Borza T，Peters Ｒ D，et al. Proteomics analysis suggestsbroad functional changes in potato leaves triggered by phosphitesand a complex indirect mode of action against Phytophthora infestans［J］. Journal of Proteomics，2013，93: 207-223.

［5］ Bashir Z，Ahmad A，Shafique S，et al. Hypersensitive response-A biophysical phenomenon of producers［J］. European Journal ofMicrobiology and Immunology，2013，3( 2) : 105-110.

［6］ van Loon L C，Pierpoint W S，Boller T. Ｒecommendations for namingplant pathogenesis-related proteins［J］. Plant Molecular BiologyＲeporter，1994，12( 3) : 245-264.

［7］ Woloshuk C P，MeulenhoV J S，Sela-Buurlage M，et al. Pathogen-induced proteins with inhibitory activity toward Phytophthorainfestans［J］. Plant Cell，1991，3: 619-628.

［8］ Sela-Buurlage M B，Ponstein A S，Bres-Vloemans S A，et al. Onlyspecific tobacco ( Nicotiana tabacum) chitinases and β-1，3-glucanasesexhibit antifungal activity［J］. Plant Physiology，1993，101: 857-863.

［9］ Dempsey D A，Shah J，Klessig D F. Salicylic acid and disease resistancein plants［J］. Critical Ｒeviews in Plant Sciences，1999，18: 547-575.

［10］ Pieterse C M，van Loon L C. Salicylic acid-independent plant defensepathways［J］. Trends in Plant Science，1999，4: 52-58.

［11］ Jun-Jun L，Abul K M，Ekramoddoullah. Ｒoot-specific expressionof a western white pine PＲ10 gene is mediated by different promoterregions in transgenic tobacco［J］. Plant Molecular Biology，2003，52: 103-120.

［12］ Chen Z Y，Brown Ｒ L，Ｒajasekaran K，et al. Identification of amaize kernel pathogenesis-related protein and evidence for its involvementin resistance to Aspergillus flavus infection and aflatoxinproduction［J］. Phytopathology，2006，96( 1) : 87-95.

［13］Ｒakwal Ｒ，Agrawal G K，Yonekura M. Light-dependent inductionOsPＲ10 in rice ( Oryza sativa L. ) seedlings by the global stresssignaling molecule jasmonic acid and protein phosphatase 2A inhibitors［J］. Plant Science，2001，161: 469-479.

［14］Ｒyu H S，Han M，Lee S K，et al. A comprehensive expression analysisof the WＲKY gene superfamily in rice plants during defenseresponse［J］. Plant Cell Ｒeports，2006，25: 836-847.

［15］ Hwang S，Lee I A，Yie S W，et al. Identification of an OsPＲ10apromoter region responsive to salicylic acid［J］. Planta，2008，227( 5) : 1141-1150.

［16］ Malnoy M，Venisse J S，Ｒeynoird J P，et al. Activation of threepathogen-inducible promoters of tobacco in transgenic pear ( Pyruscommunis L. ) after abiotic and biotic elicitation［J］. Planta，4 期孟凡姗等: 大豆GmPＲ10 基因启动子的克隆及序列分析5732003，216: 802-814.

［17］ Hong J K，Lee S C，Hwang B K. Activation of pepper basic PＲ-1gene promoter during defense signaling to pathogen，abiotic andenvironmental stresses［J］. Gene，2005，356: 169-180.

［18］ Li Y F，Zhu Ｒ，Xu P. Activation of the gene promoter of barley β-1，3-glucanase isoenzyme GIII is salicylic acid ( SA) -dependent intransgenic rice plants［J］. Journal of Plant Ｒesearch，2005，118:215-221.

［19］ Xu P F，Jiang L Y，Wu J J，et al. Isolation and characterizationof a pathogenesis-related protein 10 gene ( GmPＲ10) with inducedexpression in soybean ( Glycine max) during infection with Phytophthorasojae［J］. Molecular Biology Ｒeporter，2014，41 ( 8 ) :4899-4909.

［20］张伟，谢甫绨，曹萍，等．大豆叶片DNA 提取方法的比较［J］．大豆科学，2007，26 ( 1) : 60-65． ( Zhang W，Xie F T，Cao P.Comparative studies on extraction of genome DNA from soybeanleaf［J］. Soybean Science，2007，26( 1) : 60-65. )

［21］ Sambrook J，Fristch E F，Mantiatis T. Molecular clonging: A laboratorymanual: 2 ed［M］. Beijing: Science Press，2002.

［22］ Higo K，Ugawa Y，Iwamoto M. Plant cis-acting regulatory DNA elements(PLACE) database［J］. Nucleic Acids Ｒesearch，1999，27( 1) : 297-300.

［23］ Prestridge D S. Signal scan: A computer program that scans DNAsequences for eukaryotic transcriptional elements［J］. ComputerApplications in the Biosciences，1997，7: 203-206.

［24］ Jefferson Ｒ A，Kavanagh T A，Bevan M W. GUS fusions: β-glucuronidaseas a sensitive and versatile gene fusion marker in higherplants［J］. EMBO Journal，1987，6: 3901-3907.

［25］ Grace M L，Chandrasekharan M B，Hall T C，et al. Sequence andspacing of TATA box elements are critical for accurate initiationfrom the beta-phaseolin promoter［J］. Journal of Biological Chemistry，2004，279: 8102-8110.

［26］ Shirsat A，Wilford N，Croy Ｒ，et al. Sequences responsible forthe tissue specific promoter activity of a pea legumin gene in tobacco［J］. Molecular and General Genetics，1989，215: 326-331.

［27］ Hartmann U，Sagasser M，Mehrtens F， et al. Differential combinatorialinteractions of cis-acting elements recognized by Ｒ2Ｒ3-MYB，BZIP，and BHLH factors control light-responsive and tissue-specific activation of phenylpropanoid biosynthesis genes［J］.Plant Molecular Biology，2005，57: 155-171.

［28］Ｒose A，Meier I，Wienand U. The tamato I-box binding factor Le-MYBI is a member of a novel class of Myb-like proteins［J］. PlantJournal，1999，20: 641-652.

［29］ Nakashima K，Fujita Y，Katsura K，et al. Transcriptional regulationof ABI3- and ABA-responsive genes including ＲD29B andＲD29A in seeds，germinating embryos，and seedlings of Arabidopsis［J］. Plant Molecular Biology，2006，60: 51-68.［30］Ｒoss E J，Stone J M，Elowsky C G，et al. Activation of the Oryzasativa non-symbiotic haemoglobin-2 promoter by the cytokinin-regulatedtranscription factor，AＲＲ1［J］. Journal of ExperimentalBotany，2004，55: 1721-1731.

［31］ Fusada N，Masuda T，Kuroda H， et al. Identification of a novel ciselementexhibiting cytokinin-dependent protein binding in vitro inthe 5’-region of NADPH-protochlorophyllide oxidoreductase gene incucumber［J］. Plant Molecular Biology，2005，59: 631-645.

［32］Ｒedman J，Whitcraft J，Johnson C， et al. Abiotic and biotic stressdifferentially stimulate as-1 element activity in Arabidopsis［J］.Plant Cell Ｒeports，2002，21: 180-185.

［33］ Xu N，Hagen G，Guilfoyle T. Multiple auxin response modules inthe soybean SAUＲ 15A promoter［J］. Plant Science，1997，126:193-201.

［34］ Yu D，Chen C，Chen Z. Evidence for an important role of WＲKYDNA binding proteins in the regulation of NPＲ1 gene expression［J］. Plant Cell，2001，13: 1527-1540.

［35］ Boter M，Ｒuiz-Ｒivero O，Abdeen A， et al. Conserved MYC transcriptionfactors play a key role in jasmonate signaling both in tomatoand Arabidopsis［J］. Genes ＆ Development，2004，18:1577-1591.

［36］ Dunn M A，White A J，Vural S，et al． Identification of promoterelements in a low-temperature-responsive gene ( blt4. 9) from barley( Hordeum vulgare L. ) ［J］. Plant Molecular Biology，1998，38: 551-564.

［37］ Abe H，Urao T，Ito T， et al. Arabidopsis AtMYC2 ( bHLH) andAtMYB2 ( MYB) function as transcriptional activators in abscisicacid signaling［J］. Plant Cell，2003，15: 63-78.

［38］ Planchais S，Perennes C，Glab N，et al. Characterization of cisactingelement involved in cell cycle phase-independent activationof Arath; CycB1; 1 transcription and identification of putative regulatoryproteins［J］. Plant Molecular Biology，2002，50: 111-127.［39］ Ericson M L，Muren E，Gustavsson H O，et al. Analysis of thepromoter region of napin genes from Brassica napus demonstratesbinding of nuclear protein in vitro to a conserved sequence motif［J］. European Journal of Biochemistry，1991，197: 741-746.［40］ Wenkel S，Turck F，Singer K， et al. CONSTANS and the CCAATBox binding complex share a functionally important domain and interactto regulate flowering of Arabidopsis［J］. Plant Cell，2006，18: 2971-2984.［41］ Kropat J，Tottey S，Birkenbihl Ｒ P， et al. A regulator of nutritionalcopper signaling in Chlamydomonas is an SBP domain proteinthat recognizes the GTAC core of copper response element［J］.Proceedings of the National Academy of Sciences of the UnitedStates of America，2005，102: 18730-18735.［42］ Hartmann U，Sagasser M，Mehrtens F， et al. Differential combinatorialinteractions of cis-acting elements recognized by Ｒ2Ｒ3-MYB，BZIP，and BHLH factors control light-responsive and tissue-specific activation of phenylpropanoid biosynthesis genes［J］.Plant Molecular Biology，2005，57: 155-171.

［43］ Nishiuchi T，Shinshi H，Suzuki K. Ｒapid and transient activationof transcription of the EＲF3 gene by wounding in tobacco leaves:Possible involvement of NtWＲKYs and autorepression［J］. Journalof Biological Chemistry，2004，279: 55355-55361.

［44］ Zhang Z L，Xie Z，Zou X，et al. A rice WＲKY gene encodes atranscriptional repressor of the gibberellin signaling pathway in aleuronecells［J］. Plant Physiology，2004，134: 1500-1513.

［45］ Valerie Ｒ，Michael K，Peter O. Transcriptional regulation of geneexpression in C. elegans［J］. WormBook，2013，4( 6) : 1-34.

［46］ Lee T I，Young Ｒ A. Transcriptional regulation and its misregulationin disease［J］. Cell，2013，152( 6) : 1237-1251.

［47］ Dong X． SA， JA， ethylene， and disease resistancein plants［J］． CurrentOpinion in Plant Biology，1998，( 1) : 316-323.

［48］ Chai C Y，Lin Y L，Shen D Y，et al. Identification and functionalcharacterization of the soybean GmaPPO12 promoter conferringPhytophthora sojae induced expression［J］. PLoS One，2013，6( 8) : e67670.

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备注/Memo

基金项目: 国家自然科学基金( 31171577，31101167) ; 黑龙江省杰出青年基金( JC201308) ; 长江后备支持计划; 龙江学者基金; 哈尔滨市科技创新项目( 2012ＲFQXN011，2012ＲFXXN019) 。第一作者简介: 孟凡姗( 1991-) ，女，硕士，主要从事大豆抗病遗传育种研究。通讯作者: 张淑珍( 1972-) ，女，博士，博导，主要从事大豆抗病遗传育种研究。E-mail: zhangshuzhen@ neau. edu. cn。

更新日期/Last Update: 2016-08-22