马铃薯晚疫病菌RxLR效应因子RD24基因克隆及其PVX表达载体构建与鉴定
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摘要
马铃薯晚疫病病原菌致病疫霉菌(Phytophthora infestans)在侵入寄主细胞的同时分泌效应因子(Effector),该类蛋白在晚疫病致病过程中发挥着关键作用,是抑制植物免疫的重要因素。PITG_RD24是马铃薯晚疫病菌位于细胞核核的一个重要效应因子。为了快速鉴定效应因子RD24在马铃薯中瞬时表达的状况及抗性情况,构建效应子RD24的PVX表达载体是十分必要的。本研究利用PCR、酶切、分子克隆等生物学技术,将RD24片段连接插入至PVX (pGR106)载体中,然后用热击法将连接产物转化至大肠杆菌DH10B感受态细胞中。通过PCR筛选、质粒提取、测序比对,结果表明成功获得含有目的表达载体pGR106·RD24的质粒,可为下一步筛选马铃薯广谱抗病基因研究提供必备载体。
The pathogen Phytophthora infestans, which causes potato late blight, delivers effectors inside the living host cells, the effector proteins play an important role for the infection of the pathogen, and are the critical reasons for the pathogen to suppress plant immunity. PITG_RD24 is one of the important effectors of P.infestans which is located in the nucleus of plant cell. To identify the transient expression and resistance of RD24 effector in potato, it is necessary to construct the expression vector of PVX·RD24(pGR106·RD24). In this study, the fragment of RD24 is inserted to empty vector of pGR106 by the methods of PCR, restriction enzyme digestion and molecular cloning. Then the connected product and plasmid is transformed into DH10 B Agrobacterium tumefaciens strains by heat shock method. The expression vector of pGR106 · RD24 is successfully obtained after PCR screening, plasmid extraction, contrasting sequence, and the study can provide the necessary vector for screening potato resistant genes.
The pathogen Phytophthora infestans, which causes potato late blight, delivers effectors inside the living host cells, the effector proteins play an important role for the infection of the pathogen, and are the critical reasons for the pathogen to suppress plant immunity. PITG_RD24 is one of the important effectors of P.infestans which is located in the nucleus of plant cell. To identify the transient expression and resistance of RD24 effector in potato, it is necessary to construct the expression vector of PVX·RD24(pGR106·RD24). In this study, the fragment of RD24 is inserted to empty vector of pGR106 by the methods of PCR, restriction enzyme digestion and molecular cloning. Then the connected product and plasmid is transformed into DH10 B Agrobacterium tumefaciens strains by heat shock method. The expression vector of pGR106 · RD24 is successfully obtained after PCR screening, plasmid extraction, contrasting sequence, and the study can provide the necessary vector for screening potato resistant genes.
引文
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[21] Chung S M, Frankman E L, Tzfira T. A versatile vector system formultiple gene expression in plants[J].Trends in Plant Science,2005,10(8):357-361.
[2] Birch P R J, Bryan G, Fenton B, et al. Crops that feed the world.Potato:are the trends of increased global production sustainable?[J].Food Security,2012,4:477-508.
[3]孙楠楠.大豆疫霉3-磷酸磷脂酰肌醇在致病过程中的功能分析[D].南京:南京农业大学,2013.
[4] Haas B J, Kamoun S, Zody M C, et al. Genome sequence andanalysis of the Irish potato famine pathogen Phytophthora infestans[J].Nature,2009,461(7262):393-8.
[5] Dou D, Kale D, Liu T, et al. Different domains of Phytophthorasojae effector Avr4/6 are recognized by soybean resistance genesRps 4 and Rps 6[J].Molecular Plant-Microbe Interactions,2010,23:425-435.
[6] Boevink P C, Wang X, Mclellan H, et al. A Phytophthora infestansRXLR effoctor targets plant PP1c isoforms that potato late blightdisease[J].Nature Communications,2016,7:10311.
[7]杨凤萍,王宏芝,陈绪清,等.利用病毒载体在烟草中瞬时表达融合HBsAg基因[J].西北植物学报,2008,28(2):217-222.
[8]曲静,郭兴启,慈晓燕,等.马铃薯X病毒分子生物学研究进展及其作为表达载体的应用[J].中国病毒学,2003,18(1):87-92.
[9] Chung S M, Frankman E L, Tzfira T. A versatile vector system formultiple gene expression in plants[J].Trends in Plant Science,2005,10(8):357-361.
[10] Berrow N S, Alderton D, Sainsbury S, et al. A versatile ligation-independent cloning method suitable for high-throughputexpression screening applications[J].Nucleic Acids Research,2007,35(6):e45.
[11] Wise M G, Siragusa G R. Quantitative analysis of the intestinalbacterial community in one-to three-week-old commercially rearedbroiler chickens fed conventional or antibiotic-free vegetable-baseddiets[J].Journal of Applied Microbiology,2007,102(4):1138.
[12] Chapman S, Hills G, Watts J, et al. Mutational analysis of the coatprotein gene of potato virus X:effects on virion morphology andviral pathogenicity[J].Virology,1992,191(1):223-230.
[13] Danan S, Veyrieras J B, Lefebvre V. Construction of a potatoconsensus map and QTL meta-analysis offer new insights into thegenetic architecture of late blight resistance and plant maturity traits.[J].Bmc Plant Biology,2011,11(1):16.
[14]牛少芳.烟草坏死病毒A外壳蛋白的核质穿梭及其功能分析[D].北京:中国农业大学,2013.
[15] Boevink P C, Wang X, Mclellan H, et al. A Phytophthora infestansRXLR effector targets plant PP1c isoforms that promote late blightdisease[J].Nature Communications,2016,7:10311.
[16]王晓丹.马铃薯晚疫病菌RxLR效应蛋白与寄主蛋白互作促进侵染的机制研究[D].哈尔滨:东北农业大学,2015.
[17] King S R, Mclellan H, Boevink P C, et al. Phytophthora infestansRXLR effector PexRD2 interacts with host MAPKKKεto suppressplant immune signaling[J].Plant Cell,2014,26(3):1345.
[18] Baloglu M C, Battal A, Aydin G, et al. Vector constructionstrategies for transformation of wheat plant[J].Journal of Animal&Plant Sciences,2013,23(3):906-912.
[19]林春晶,韦正乙,蔡勤安,等.几种植物转基因表达载体的构建方法[J].生物技术,2008,18(5):84-87.
[20] Kuhnlein U, Linn S, Arber W. Host Specificity of DNA Producedby Escherichia coli, XI. In vitro Modification of Phage fdReplicative Form[J].Proc Natl Acad Sci U S A,1969,63(2):556-562.
[21] Chung S M, Frankman E L, Tzfira T. A versatile vector system formultiple gene expression in plants[J].Trends in Plant Science,2005,10(8):357-361.