三种单子叶植物叶组织中GWSF诱导转录特性
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摘要
理想病原诱导型启动子的应用在植物抗病基因工程中非常重要。本研究以高粱、玉米和小麦为材料,检测人工病原诱导型启动子GWSF在单子叶植物叶组织中的诱导转录特性,寻找理想病原诱导型启动子。用GWSF替代pBI121中调控gus基因的CaMV35S启动子构建重组质粒,导入农杆菌(Agrobacterium tumefaciens) GV3101;通过农杆菌渗入法转化高粱、玉米和小麦12 h后,用水杨酸,青枯菌,疫霉菌孢子诱导处理12 h,本底表达用无菌水处理12 h;最后用GUS染色法和实时荧光定量PCR (real-time quantitative PCR, qPCR)检测GWSF的转录特性。GUS染色结果显示:GWSF在三种植物中都具有本底低、受水杨酸,青枯菌,疫霉菌孢子诱导的特性。设CaMV35S的转录活性为1,qPCR结果为:GWSF在高粱中本底水平为0.67,受水杨酸,青枯菌,疫霉菌孢子诱导时,转录活性明显升高,分别为:2.53、0.87、7.33;玉米中本底水平为0.11,受水杨酸,青枯菌,疫霉菌孢子诱导时,转录活性分别为:1.92、0.19、2.06;小麦中本底水平为0.13,受水杨酸,青枯菌,疫霉菌孢子诱导时,转录活性分别为:0.69、0.45、1.16。结果表明,GWSF在三种单子叶植物叶组织中具有本底低、诱导因子广、诱导活性高的特性,是较理想的人工病原诱导型启动子,可应用于单子叶植物转基因抗病育种。
The application of ideal pathogen inducible promoter is very important in plant disease resistance genetic engineering. In this study, sorghum, maize and wheat were used as materials to evaluate the induced transcriptional properties of GWSF promoter in monocotyledonous leaf tissues, and to find the ideal pathogen-inducible promoter. GWSF was used to replace the CaMV35 S promoter which regulated gus gene in the pBI121 in order to construct recombinant plasmid. Then, the recombinant plasmid was introduced into Agrobacterium tumefaciensus GV3101. The sorghum, maize and wheat were transformed by agroinfiltration for 12 hours. Then, the salicylic acid, Ralstonia solanacearum and Phytophthora spores were induced for 12 hours, and the background expression was treated with sterile water for 12 hours. Finally, the transcriptional properties of GWSF were evaluated by GUS staining and real-time quantitative quantitative(qPCR). GUS staining showed that GWSF had the properties of low background and induction by salicylic acid, Ralstonia solanacearum and Phytophthora spores. With the transcriptional activity of the CaMV35 S be normalized to one, the qPCR results showed that the background level of GWSF in sorghum was 0.67, and the relative transcriptal activity significantly increased to 2.53, 0.87, 7.33 after being induced by salicylic acid, Ralstonia solanacearum and Phytophthora spores, respectively. Meanwhile, the background level of GWSF in maize was 0.11, and relative transcriptal activity were 1.92, 0.19 and 2.06. And the background level was 0.13 and relative transcriptal activity were 0.69, 0.45 and 1.16 in wheat. The results indicated that GWSF had the characteristics of low background, wide inducing factors and high induced activity in the leaf tissues of three monocotyledons. It might be an ideal promoter for artificial pathogen induction and could be apply to transgenic disease resistance breeding in monocotyledonous plants.
The application of ideal pathogen inducible promoter is very important in plant disease resistance genetic engineering. In this study, sorghum, maize and wheat were used as materials to evaluate the induced transcriptional properties of GWSF promoter in monocotyledonous leaf tissues, and to find the ideal pathogen-inducible promoter. GWSF was used to replace the CaMV35 S promoter which regulated gus gene in the pBI121 in order to construct recombinant plasmid. Then, the recombinant plasmid was introduced into Agrobacterium tumefaciensus GV3101. The sorghum, maize and wheat were transformed by agroinfiltration for 12 hours. Then, the salicylic acid, Ralstonia solanacearum and Phytophthora spores were induced for 12 hours, and the background expression was treated with sterile water for 12 hours. Finally, the transcriptional properties of GWSF were evaluated by GUS staining and real-time quantitative quantitative(qPCR). GUS staining showed that GWSF had the properties of low background and induction by salicylic acid, Ralstonia solanacearum and Phytophthora spores. With the transcriptional activity of the CaMV35 S be normalized to one, the qPCR results showed that the background level of GWSF in sorghum was 0.67, and the relative transcriptal activity significantly increased to 2.53, 0.87, 7.33 after being induced by salicylic acid, Ralstonia solanacearum and Phytophthora spores, respectively. Meanwhile, the background level of GWSF in maize was 0.11, and relative transcriptal activity were 1.92, 0.19 and 2.06. And the background level was 0.13 and relative transcriptal activity were 0.69, 0.45 and 1.16 in wheat. The results indicated that GWSF had the characteristics of low background, wide inducing factors and high induced activity in the leaf tissues of three monocotyledons. It might be an ideal promoter for artificial pathogen induction and could be apply to transgenic disease resistance breeding in monocotyledonous plants.
引文
Dey N.,Sa rkar S.,Acharya S.,and Maiti I.B.,2015,Synthetic promoters in planta,Planta,242(5):1077-1094
Fan B.Y.,Gao S.P.,Hou X.G.,Xu H.W.,Shi G.A.,and Kong X.S.,2011,Molecular cloning of promoter of AP3 gene from Arabidopsis thaliana(Ecotype Col)and construction of plant expression vector,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),30(1):21-26(范丙友,高水平,侯小改,胥华伟,史国安,孔祥生,2011,Col生态型拟南芥AP3基因启动子克隆及植物表达载体构建,基因组学与应用生物学,30(1):21-26)
Huang Z.C.,Peng S.,Li H.,and Zeng F.H.,2017,Transcriptional properties of eight synthetic pathogen-inducible promoters in transgenic Arabidopsis thaliana,Biol.Plant,61(2):389-393
Huang Z.C.,and Li H.,2018,Transcriptional properties and transcriptional activities to ethylene of a synthetic pathogen-inducible promoter GWSF in Arabidopsis thaliana,Zhiwu Shengli Xuebao(Plant Physiology Journal),54(1):121-126(黄真池,李恒,2018,拟南芥中人工启动子GWSF转录特性及乙烯诱导活性分析,植物生理学报,54(1):121-126)
Jefferson R.A.,Kavanagh T.A.,and Bevan M.W.,1987,GUS fusions:β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants,EMBO J.,6(13):3901-3907
Kinkema M.,Geijskes R.J.,Shand K.,Coleman H.D.,De Lucca P.C.,Palupe A.,Harrison M.D.,Jepson I.,Dale J.L.,and Sainz M.B.,2014,An improved chemically inducible gene switch that functions in the monocotyledonous plant sugar cane,Plant Mol.Biol.,84(4-5):443-454
Lehmeyer M.,Kanofsky K.,Hanko E.K.,Ahrendt S.,Wehrs M.,Machens F.,and Hehl R.,2016,Functional dissection of a strong and specific microbe-associated molecular patternresponsive synthetic promoter,Plant Biotechnol.J.,14(1):61-71
Liu W.S.,and Jr Stewart Neal C.,2016,Plant synthetic promoters and transcription factors,Curr.Opin.Biotechnol.,37:36-44
Liu W.S.,Mazarei M.,Rudis M.R.,Fethe M.H.,and Jr Stewart Neal C.,2011,Rapid in vivo analysis of synthetic promoters for plant pathogen phytosensing,BMC Biotechnol.,11(1):108
Mohr T.J.,Mammarella N.D.,Hoff T.,Woffenden B.J.,Jelesko J.G.,and Mcdowell J.M.,2010,The Arabidopsis downy mildew resistance gene RPP8 is induced by pathogens and salicylic acid and is regulated by W box cis elements,Mol.Plant Microbe Interact.,23(10):1303-1315
Peng S.,Huang Z.C.,Ouyang L.J.,and Zeng F.H.,2011,Research progress of artificial promoter in plant genetic engineering,Zhiwu Shengli Xuebao(Plant Physiology Journal),47(2):141-146(彭舒,黄真池,欧阳乐军,曾富华,2011,植物基因工程中人工启动子的研究进展,植物生理学报,47(2):141-146)
Pfaffl M.W.,2001,A new mathematical model for relative quantification in real-time RT-PCR,Nucleic Acids Res.,29(9):e45
Qiu R.,Tao G.,Li Q.K.,Qiu Y.B.,and Liu Z.Y.,2009,Transient gene expression mediated by agroinfiltration and its appli cation,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),7(5):1032-1039(邱礽,陶刚,李奇科,邱又彬,刘作易,2009,农杆菌渗入法介导的基因瞬时表达技术及应用,分子植物育种,7(5):1032-1039)
Wei K.F.,Liu Y.P.,Lin Z.Y.,Yang Y.F.,Zhang Z.H.,and Jia W.S.,2008,Problems and solutions in Agrobacterium tumefaciens-mediated genetic transformation of monocotyledons,Zhiwuxue Tongbao(Chinese Builetin of Botany),25(4):491-496(魏开发,刘逸萍,林子英,杨雅芳,张泽宏,贾文锁,2008,农杆菌介导单子叶植物遗传转化问题与对策,植物学通报,25(4):491-496)
Sahoo D.K.,Sarkar S.,Raha S.,Maiti I.B.,and Dey N.,2014,Comparative analysis of synthetic DNA promoters for highlevel gene expression in plants,Planta,240(4):855-875
Schlabach R.M.,Hu J.K.,Li M.M.,and Elledge J.S.,2010,Synthetic design of strong promoters,Proc.Natl.Acad.Sci.USA,107(6):2538-2543
Yang J.W.,Cheng X.L.,and Ge Z.L.,2014,Cloning and characterization of leaf specific promoter from potato(Solanum tuberosum L.)and its application in human IL12 expression vector construction,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),33(4):875-881(杨加伟,程小玲,葛正龙,2014,马铃薯叶片特异性启动子克隆分析及其驱动的人白细胞介素-12表达载体构建,基因组学与应用生物学,33(4):875-881)
Yang Y.,Li R.,and Qi M.,2000,In vivo analysis of plant promoters and transcription factors by agroinfiltration of tobacco leaves,Plant J.,22(6):543-551
Fan B.Y.,Gao S.P.,Hou X.G.,Xu H.W.,Shi G.A.,and Kong X.S.,2011,Molecular cloning of promoter of AP3 gene from Arabidopsis thaliana(Ecotype Col)and construction of plant expression vector,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),30(1):21-26(范丙友,高水平,侯小改,胥华伟,史国安,孔祥生,2011,Col生态型拟南芥AP3基因启动子克隆及植物表达载体构建,基因组学与应用生物学,30(1):21-26)
Huang Z.C.,Peng S.,Li H.,and Zeng F.H.,2017,Transcriptional properties of eight synthetic pathogen-inducible promoters in transgenic Arabidopsis thaliana,Biol.Plant,61(2):389-393
Huang Z.C.,and Li H.,2018,Transcriptional properties and transcriptional activities to ethylene of a synthetic pathogen-inducible promoter GWSF in Arabidopsis thaliana,Zhiwu Shengli Xuebao(Plant Physiology Journal),54(1):121-126(黄真池,李恒,2018,拟南芥中人工启动子GWSF转录特性及乙烯诱导活性分析,植物生理学报,54(1):121-126)
Jefferson R.A.,Kavanagh T.A.,and Bevan M.W.,1987,GUS fusions:β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants,EMBO J.,6(13):3901-3907
Kinkema M.,Geijskes R.J.,Shand K.,Coleman H.D.,De Lucca P.C.,Palupe A.,Harrison M.D.,Jepson I.,Dale J.L.,and Sainz M.B.,2014,An improved chemically inducible gene switch that functions in the monocotyledonous plant sugar cane,Plant Mol.Biol.,84(4-5):443-454
Lehmeyer M.,Kanofsky K.,Hanko E.K.,Ahrendt S.,Wehrs M.,Machens F.,and Hehl R.,2016,Functional dissection of a strong and specific microbe-associated molecular patternresponsive synthetic promoter,Plant Biotechnol.J.,14(1):61-71
Liu W.S.,and Jr Stewart Neal C.,2016,Plant synthetic promoters and transcription factors,Curr.Opin.Biotechnol.,37:36-44
Liu W.S.,Mazarei M.,Rudis M.R.,Fethe M.H.,and Jr Stewart Neal C.,2011,Rapid in vivo analysis of synthetic promoters for plant pathogen phytosensing,BMC Biotechnol.,11(1):108
Mohr T.J.,Mammarella N.D.,Hoff T.,Woffenden B.J.,Jelesko J.G.,and Mcdowell J.M.,2010,The Arabidopsis downy mildew resistance gene RPP8 is induced by pathogens and salicylic acid and is regulated by W box cis elements,Mol.Plant Microbe Interact.,23(10):1303-1315
Peng S.,Huang Z.C.,Ouyang L.J.,and Zeng F.H.,2011,Research progress of artificial promoter in plant genetic engineering,Zhiwu Shengli Xuebao(Plant Physiology Journal),47(2):141-146(彭舒,黄真池,欧阳乐军,曾富华,2011,植物基因工程中人工启动子的研究进展,植物生理学报,47(2):141-146)
Pfaffl M.W.,2001,A new mathematical model for relative quantification in real-time RT-PCR,Nucleic Acids Res.,29(9):e45
Qiu R.,Tao G.,Li Q.K.,Qiu Y.B.,and Liu Z.Y.,2009,Transient gene expression mediated by agroinfiltration and its appli cation,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),7(5):1032-1039(邱礽,陶刚,李奇科,邱又彬,刘作易,2009,农杆菌渗入法介导的基因瞬时表达技术及应用,分子植物育种,7(5):1032-1039)
Wei K.F.,Liu Y.P.,Lin Z.Y.,Yang Y.F.,Zhang Z.H.,and Jia W.S.,2008,Problems and solutions in Agrobacterium tumefaciens-mediated genetic transformation of monocotyledons,Zhiwuxue Tongbao(Chinese Builetin of Botany),25(4):491-496(魏开发,刘逸萍,林子英,杨雅芳,张泽宏,贾文锁,2008,农杆菌介导单子叶植物遗传转化问题与对策,植物学通报,25(4):491-496)
Sahoo D.K.,Sarkar S.,Raha S.,Maiti I.B.,and Dey N.,2014,Comparative analysis of synthetic DNA promoters for highlevel gene expression in plants,Planta,240(4):855-875
Schlabach R.M.,Hu J.K.,Li M.M.,and Elledge J.S.,2010,Synthetic design of strong promoters,Proc.Natl.Acad.Sci.USA,107(6):2538-2543
Yang J.W.,Cheng X.L.,and Ge Z.L.,2014,Cloning and characterization of leaf specific promoter from potato(Solanum tuberosum L.)and its application in human IL12 expression vector construction,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),33(4):875-881(杨加伟,程小玲,葛正龙,2014,马铃薯叶片特异性启动子克隆分析及其驱动的人白细胞介素-12表达载体构建,基因组学与应用生物学,33(4):875-881)
Yang Y.,Li R.,and Qi M.,2000,In vivo analysis of plant promoters and transcription factors by agroinfiltration of tobacco leaves,Plant J.,22(6):543-551