小麦和大麦GER4c基因启动子的分离及其锈菌诱导特性分析
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摘要
病原菌诱导型启动子在植物-病原菌互作研究及基因工程育种中具有重要的利用价值,可根据抗病性的需求调控目的基因的表达。为挖掘小麦来源的锈菌诱导型启动子,为小麦抗条锈病的理论研究及育种应用提供基础,本研究分离了大麦类萌发素蛋白GER4c基因及其小麦同源基因的启动子,利用报告基因GFP表达载体在二穗短柄草中的异源表达,分析了启动子功能。发现小麦及大麦GER4c基因启动子表达模式相同,驱动报告基因在叶片中的表达量最高,在叶鞘、茎秆和小穗中显著降低。两个启动子均表现为锈菌诱导型启动子,转基因短柄草在接种锈菌小种F-Fl后,RT-PCR检测GFP在叶片中的表达量显著上调,显微镜下绿色荧光蛋白信号明显增强。研究结果为开展麦类作物抗条锈病相关研究提供了作物自身来源的诱导型启动子。
Pathogen-inducible promoter is a useful tool for studying plant-pathogen interaction and for molecular breeding,because such a promoter confers specific gene expression only when the plant pathogen presents. In order to identify rust pathogen inducible promoter in wheat and therefore provide basis for wheat rust-resistance research and breeding, we isolated two promoters of the Germin-like protein GER4 c gene, one from barley and another from wheat. Expression vectors with the GFP fusion tag have been constructed and transformed into Brachypodium distachyon. Same expression pattern was found between the GER4 c promoters of wheat and barley, of that the expression level of GFP was the highest in leaves and significantly lower in leaf sheaths, stems and spikelets. It was also found that both the wheat and barley GER4 c promoters are rust pathogen inducible. When infected with Puccinia brachypodii isolate F-Fl, both the cDNA expression level and the fluorescent signal of the reporter gene GFP were significantly up-regulated in the leaves of transgenic Brachypodium. This study provides a rust pathogen inducible promoter to facilitate basic research and rust resistance breeding in wheat.
Pathogen-inducible promoter is a useful tool for studying plant-pathogen interaction and for molecular breeding,because such a promoter confers specific gene expression only when the plant pathogen presents. In order to identify rust pathogen inducible promoter in wheat and therefore provide basis for wheat rust-resistance research and breeding, we isolated two promoters of the Germin-like protein GER4 c gene, one from barley and another from wheat. Expression vectors with the GFP fusion tag have been constructed and transformed into Brachypodium distachyon. Same expression pattern was found between the GER4 c promoters of wheat and barley, of that the expression level of GFP was the highest in leaves and significantly lower in leaf sheaths, stems and spikelets. It was also found that both the wheat and barley GER4 c promoters are rust pathogen inducible. When infected with Puccinia brachypodii isolate F-Fl, both the cDNA expression level and the fluorescent signal of the reporter gene GFP were significantly up-regulated in the leaves of transgenic Brachypodium. This study provides a rust pathogen inducible promoter to facilitate basic research and rust resistance breeding in wheat.
引文
[1]李田,孙景宽,刘京涛.植物启动子研究进展[J].生物技术通报,2015,31(2):18-25
[2]孙芳芳,宋洪元.植物诱导型启动子研究进展[J].南方园艺,2014,25(2):51-56
[3]李杰,张福城,王文泉,等.高等植物启动子的研究进展[J].生物技术通讯,2006,17(4):658-661
[4]王淼,王旭静,唐巧玲,等.高等植物绿色组织特异表达启动子研究进展[J].中国农业科技导报,2010,12(2):33-37
[5]Cai M,Qiu D,Yuan T,et al.Identification of novel pathogen-responsive cis-elements and their binding proteins in the promoter of OsWRKY13,a gene regulating rice disease resistance[J].Plant Cell&Environment,2008,31(1):86-96
[6]Chujo T,Sugioka N,Masuda Y,et al.Promoter analysis of the elicitor-induced WRKY gene OsWRKY53,which is involved in defense responses in rice[J].Biosci Biotechnol Biochem,2009,73(8):1901-1904
[7]李甜甜,李丽丽,陈利红,等.水稻茉莉酸甲酯和白叶枯病菌诱导型启动子的克隆及功能鉴定[J].分子植物育种,2018,16(3):689-695
[8]Petitot AS,Barsalobres-Cavallari C,Ramiro D,et al.Promoter analysis of the WRKY transcription factors CaWRKY1a and CaWRKY1b homoeologous genes in coffee(Coffea arabica)[J].Plant Cell Reports,2013,32(8):1263-1276
[9]Himmelbach A,Liu L,Zierold U,et al.Promoters of the Barley Germin-Like GER4 gene cluster enable strong transgene expression in response to pathogen attack[J].American Society of Plant Biologists,2010,22(3):937-952
[10]Boni R,Chauhan H,Hensel G,et al.Pathogen-inducible Ta-Lr34res expression in heterologous barley confers disease resistance without negative pleiotropic effects[J].Plant Biotechnology Journal,2018,16(1):245-253
[11]Himmelbach A,Zierold U,Hensel G,et al.A set of modular binary vectors for transformation of cereals[J].Plant Physiology,2007,145(4):1192-1200
[12]Barbieri M,Marcel TC,Niks RE.Host status of false brome grass to the leaf rust fungus Puccinia brachypodii and the stripe rust fungus P.striiformis[J].Plant Disease,2011,95(11):1339-1345
[13]Bragg JN,Wu J,Gordon SP,et al.Generation and characterization of the Western Regional Research Center Brachypodium T-DNA insertional mutant collection[J].Plos One,2012,7(9):e41916
[14]李文艳.二穗短柄草中VRT2-like基因的克隆及初步的功能分析[D].泰安:山东农业大学,2011:35-35
[15]Lescot M,Déhais P,Thijs G,et al.PlantCARE,a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences[J].Nucleic Acids Research,2002,30(1):325-327
[16]Denance N,Sanchez-Vallet A,Goffner D,et al.Disease resistance or growth:the role of plant hormones in balancing immune responses and fitness costs[J].Frontiers in Plant Science,2013,4(1):7-30
[17]Avni R,Nave M,Barad O,et al.Wild emmer genome architecture and diversity elucidate wheat evolution and domestication[J].Science,2017,357(6346):93-97
[18]Mascher M,Gundlach H,Himmelbach A,et al.A chromosome conformation capture ordered sequence of the barley genome[J].Nature,2017,544(7651):427-433
[19]Scholthof KBG,Irigoyen S,Catalan P,et al.Brachypodium:A monocot grass model genus for plant biology[J/OL].The Plant Cell.https://doi.org/10.1105/tpc.18.00083,2018
[20]Qin Z,Wu J,Geng S,et al.Regulation of FT splicing by an endogenous cue in temperate grasses[J].Nature Communications,2017(8):14320
[21]Ni F,Qi J,Hao Q,et al.Wheat Ms2 encodes for an orphan protein that confers male sterility in grass species[J].Nature Communications,2017,8:15121
[2]孙芳芳,宋洪元.植物诱导型启动子研究进展[J].南方园艺,2014,25(2):51-56
[3]李杰,张福城,王文泉,等.高等植物启动子的研究进展[J].生物技术通讯,2006,17(4):658-661
[4]王淼,王旭静,唐巧玲,等.高等植物绿色组织特异表达启动子研究进展[J].中国农业科技导报,2010,12(2):33-37
[5]Cai M,Qiu D,Yuan T,et al.Identification of novel pathogen-responsive cis-elements and their binding proteins in the promoter of OsWRKY13,a gene regulating rice disease resistance[J].Plant Cell&Environment,2008,31(1):86-96
[6]Chujo T,Sugioka N,Masuda Y,et al.Promoter analysis of the elicitor-induced WRKY gene OsWRKY53,which is involved in defense responses in rice[J].Biosci Biotechnol Biochem,2009,73(8):1901-1904
[7]李甜甜,李丽丽,陈利红,等.水稻茉莉酸甲酯和白叶枯病菌诱导型启动子的克隆及功能鉴定[J].分子植物育种,2018,16(3):689-695
[8]Petitot AS,Barsalobres-Cavallari C,Ramiro D,et al.Promoter analysis of the WRKY transcription factors CaWRKY1a and CaWRKY1b homoeologous genes in coffee(Coffea arabica)[J].Plant Cell Reports,2013,32(8):1263-1276
[9]Himmelbach A,Liu L,Zierold U,et al.Promoters of the Barley Germin-Like GER4 gene cluster enable strong transgene expression in response to pathogen attack[J].American Society of Plant Biologists,2010,22(3):937-952
[10]Boni R,Chauhan H,Hensel G,et al.Pathogen-inducible Ta-Lr34res expression in heterologous barley confers disease resistance without negative pleiotropic effects[J].Plant Biotechnology Journal,2018,16(1):245-253
[11]Himmelbach A,Zierold U,Hensel G,et al.A set of modular binary vectors for transformation of cereals[J].Plant Physiology,2007,145(4):1192-1200
[12]Barbieri M,Marcel TC,Niks RE.Host status of false brome grass to the leaf rust fungus Puccinia brachypodii and the stripe rust fungus P.striiformis[J].Plant Disease,2011,95(11):1339-1345
[13]Bragg JN,Wu J,Gordon SP,et al.Generation and characterization of the Western Regional Research Center Brachypodium T-DNA insertional mutant collection[J].Plos One,2012,7(9):e41916
[14]李文艳.二穗短柄草中VRT2-like基因的克隆及初步的功能分析[D].泰安:山东农业大学,2011:35-35
[15]Lescot M,Déhais P,Thijs G,et al.PlantCARE,a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences[J].Nucleic Acids Research,2002,30(1):325-327
[16]Denance N,Sanchez-Vallet A,Goffner D,et al.Disease resistance or growth:the role of plant hormones in balancing immune responses and fitness costs[J].Frontiers in Plant Science,2013,4(1):7-30
[17]Avni R,Nave M,Barad O,et al.Wild emmer genome architecture and diversity elucidate wheat evolution and domestication[J].Science,2017,357(6346):93-97
[18]Mascher M,Gundlach H,Himmelbach A,et al.A chromosome conformation capture ordered sequence of the barley genome[J].Nature,2017,544(7651):427-433
[19]Scholthof KBG,Irigoyen S,Catalan P,et al.Brachypodium:A monocot grass model genus for plant biology[J/OL].The Plant Cell.https://doi.org/10.1105/tpc.18.00083,2018
[20]Qin Z,Wu J,Geng S,et al.Regulation of FT splicing by an endogenous cue in temperate grasses[J].Nature Communications,2017(8):14320
[21]Ni F,Qi J,Hao Q,et al.Wheat Ms2 encodes for an orphan protein that confers male sterility in grass species[J].Nature Communications,2017,8:15121