东方百合(Lilium)杂交品种索邦病程相关蛋白(PR1)基因的克隆与原核表达
|
摘要
病程相关蛋白(PR)广泛参与植物对生物和非生物胁迫的应答。采用cDNA末端快速克隆技术,从东方百合(Lilium)杂交品种索邦中克隆了PR1基因,并命名为LhSorPR1。LhSorPR1基因cDNA全长870bp,5’非翻译区和3’非翻译区的长度分别为47bp和274bp。该基因的开放阅读框为549bp,编码182个氨基酸。成熟的LhSorPR1蛋白(不含信号肽)分子量为18.19kDa,等电点为5.69。采用同源建模方式构建的LhSorPR1蛋白三维结构与其模板番茄PR1蛋白p14a具有较高的相似性,说明百合PR1蛋白在结构和功能上与其他植物中的PR1蛋白具有较强的保守性。构建pCold II-LhSorPR1原核表达,经IPTG诱导后,目的蛋白在大肠杆菌中以包涵体形式成功表达。使用免疫印迹方法对表达的LhSorPR1重组蛋白进行了验证,并对带有His标签的重组蛋白成功进行了纯化。
Pathogenesis-related(PR)proteins are widely involved in plant defense against both biotic and abiotic stresses.In the present study,we characterized a PR1 protein from the oriental hyrid lily cultivar Sorbonne via rapid amplification of cDNA ends(RACE),and designated as LhSorPR1.The cDNA full-length of LhSorPR1 isolated was 870 bp,consisting a5'untranslate region(UTR)of 47 bp and a 3'UTR of 274 bp.The predicted open reading frame(ORF)of LhSorPR1 was 549 bp,encoded a protein of 182 amino acids.The mature LhSorPR1(devoid of a signal peptide)had a theoretical isoelectric point of 5.69 and a calculated molecular weight of 18.19 kDa.The three dimentional(3D)model of LhSorPR1 was bulit by homology modelling,and the bulit model showed high similarity to its template p14 a.The similarity in 3D strucutures indicated the conservation of protein function of PR1 proteins in plants.The pCold II-LhSorPR1 recombinant vector was constructed to express LhSorPR1 in E.coli,and the His_6-tagged recombinant protein was expressed as inclusion bodies in the insoluble fraction under IPTG induction.The His_6-labeled recombinant LhSorPR1 protein was verified by immunoblot,and purified by chromatography.
Pathogenesis-related(PR)proteins are widely involved in plant defense against both biotic and abiotic stresses.In the present study,we characterized a PR1 protein from the oriental hyrid lily cultivar Sorbonne via rapid amplification of cDNA ends(RACE),and designated as LhSorPR1.The cDNA full-length of LhSorPR1 isolated was 870 bp,consisting a5'untranslate region(UTR)of 47 bp and a 3'UTR of 274 bp.The predicted open reading frame(ORF)of LhSorPR1 was 549 bp,encoded a protein of 182 amino acids.The mature LhSorPR1(devoid of a signal peptide)had a theoretical isoelectric point of 5.69 and a calculated molecular weight of 18.19 kDa.The three dimentional(3D)model of LhSorPR1 was bulit by homology modelling,and the bulit model showed high similarity to its template p14 a.The similarity in 3D strucutures indicated the conservation of protein function of PR1 proteins in plants.The pCold II-LhSorPR1 recombinant vector was constructed to express LhSorPR1 in E.coli,and the His_6-tagged recombinant protein was expressed as inclusion bodies in the insoluble fraction under IPTG induction.The His_6-labeled recombinant LhSorPR1 protein was verified by immunoblot,and purified by chromatography.
引文
[1]Jones J D,Dangl J L.The plant immune system[J].Nature,2006,444:323-329.
[2]Glazebrook J.Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens[J].Annual Review of Phytopathology,2005,43:205-227.
[3]van Loon L C,Rep M,Pieterse C M J.Significance of inducible defense-related proteins in infected plants[J].Annual Review of Phytopathology,2006,44:135-162.
[4]Pereira M S,de Andrade S E M,Matos L E,et al.The pathogenesis-related protein PR-4bfromTheobroma cacao presents RNase activity,Ca2+and Mg2+dependent-DNase activity and antifungal action on Moniliophthora perniciosa[J].BMC Plant Biology,2014,14:161.
[5]Antoniw J F,Pierpoint W S.Purification of a tobacco leaf protein associated with resistance to virus infection[J].Biochemical Society Transactions,1978,6:248-250.
[6]Penninckx I A M A,Eggermont K,Terras F R G,et al.Pathogen-induced systemic activation of a plant defensin gene in Arabidopsis follows a salicylic acid-independent pathway[J].Plant Cell,1996,8:2309-2323.
[7]Liu Q P,Xue Q Z.Computational identification of novel PR-1-type genes in Oryza sativa[J].Journal of Genetics,2006,85:193-198.
[8]Niderman T,Genetet I,Bruyere T,et al.Pathogenesis-Related PR-1proteins are antifungal:isolation and characterization of three 14-kilodalton proteins of tomato and of a basic PR-1of tobacco with inhibitory activity against Phytophthora infestans[J].Plant Physiology,1995,108:17-27.
[9]Buch F,Pauchet Y,Rott M,et al.Characterization and heterologous expression of a PR-1protein from traps of the carnivorous plant Nepenthes mirabilis[J].Phytochemistry,2014,100:43-50.
[10]Schultheiss H,Dechert C,Kiraly L,et al.Functional assessment of the pathogenesis-related protein PR-1bin barley[J].Plant Science,2003,165:1275-1280.
[11]Lu S W,Friesen T L,Faris J D.Molecular characterization and genomic mapping of the pathogenesis-related protein 1(PR-1)gene family in hexaploid wheat(Triticum aestivum L.)[J].Molecular Genetics and Genomics,2011,285:485-503.
[12]Li Z T,Dhekney S A,Gray D J.PR-1gene family of grapevine:a uniquely duplicated PR-1gene from a Vitis interspecific hybrid confers high level resistance to bacterial disease in transgenic tobacco[J].Plant Cell Reports,2011,30:1-11.
[13]Hong J K,Hwang B K.Induction of enhanced disease resistance and oxidative stress tolerance by overexpression of pepper basic PR-1gene in Arabidopsis[J].Physiologia Plantarum,2005,124:267-277.
[14]Zhang Y B,Wang Y J,Meng J,et al.Development of an immunochromatographic strip test for rapid detection of lily symptomless virus[J].Journal of Virological Methods,2015,220:13-17.
[15]Zhang Y B,Wang Y J,Yang W R,et al.A rapid immunochromatographic test to detect the lily mottle virus[J].Journal of Virological Methods,2015,220:43-48.
[16]Lee J A,Choi S K,Yoon J Y,et al.Variation in the pathogenicity of lily isolates of cucumber mosaic virus[J].Plant Pathology Journal,2007,23:251-259.
[17]Liu Y H,Huang C J,Chen C Y.Evidence of induced systemic resistance against Botrytis elliptica in lily[J].Phytopathology,2008,98:830-836.
[18]Curir P,Guglieri L,Dolci M,et al.Fusaric acid production by Fusarium oxysporumf.sp lilii and its role in the lily basal rot disease[J].European Journal of Plant Pathology,2000,106:849-856.
[19]李谋强,陈君良,黄彦玮,等.不同海拔区兰州百合(Lilium davidii.var.unicolor)栽培群体遗传多样性[J].中国沙漠,2016,36(4):1029-1033.
[20]Hoegen E,Stromberg A,Pihlgren U,et al.Primary structure and tissue-specific expression of the pathogenesis-related protein PR-1bin potato[J].Molecular Plant Pathology,2002,3:329-345.
[21]Talley K,Alexov E.On the pH-optimum of activity and stability of proteins[J].Proteins,2010,78:2699-2706.
[22]Qing G L,Ma L C,Khorchid A,et al.Cold-shock induced high-yield protein production in Escherichia coli[J].Nature Biotechnology,2004,22:877-882.
[2]Glazebrook J.Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens[J].Annual Review of Phytopathology,2005,43:205-227.
[3]van Loon L C,Rep M,Pieterse C M J.Significance of inducible defense-related proteins in infected plants[J].Annual Review of Phytopathology,2006,44:135-162.
[4]Pereira M S,de Andrade S E M,Matos L E,et al.The pathogenesis-related protein PR-4bfromTheobroma cacao presents RNase activity,Ca2+and Mg2+dependent-DNase activity and antifungal action on Moniliophthora perniciosa[J].BMC Plant Biology,2014,14:161.
[5]Antoniw J F,Pierpoint W S.Purification of a tobacco leaf protein associated with resistance to virus infection[J].Biochemical Society Transactions,1978,6:248-250.
[6]Penninckx I A M A,Eggermont K,Terras F R G,et al.Pathogen-induced systemic activation of a plant defensin gene in Arabidopsis follows a salicylic acid-independent pathway[J].Plant Cell,1996,8:2309-2323.
[7]Liu Q P,Xue Q Z.Computational identification of novel PR-1-type genes in Oryza sativa[J].Journal of Genetics,2006,85:193-198.
[8]Niderman T,Genetet I,Bruyere T,et al.Pathogenesis-Related PR-1proteins are antifungal:isolation and characterization of three 14-kilodalton proteins of tomato and of a basic PR-1of tobacco with inhibitory activity against Phytophthora infestans[J].Plant Physiology,1995,108:17-27.
[9]Buch F,Pauchet Y,Rott M,et al.Characterization and heterologous expression of a PR-1protein from traps of the carnivorous plant Nepenthes mirabilis[J].Phytochemistry,2014,100:43-50.
[10]Schultheiss H,Dechert C,Kiraly L,et al.Functional assessment of the pathogenesis-related protein PR-1bin barley[J].Plant Science,2003,165:1275-1280.
[11]Lu S W,Friesen T L,Faris J D.Molecular characterization and genomic mapping of the pathogenesis-related protein 1(PR-1)gene family in hexaploid wheat(Triticum aestivum L.)[J].Molecular Genetics and Genomics,2011,285:485-503.
[12]Li Z T,Dhekney S A,Gray D J.PR-1gene family of grapevine:a uniquely duplicated PR-1gene from a Vitis interspecific hybrid confers high level resistance to bacterial disease in transgenic tobacco[J].Plant Cell Reports,2011,30:1-11.
[13]Hong J K,Hwang B K.Induction of enhanced disease resistance and oxidative stress tolerance by overexpression of pepper basic PR-1gene in Arabidopsis[J].Physiologia Plantarum,2005,124:267-277.
[14]Zhang Y B,Wang Y J,Meng J,et al.Development of an immunochromatographic strip test for rapid detection of lily symptomless virus[J].Journal of Virological Methods,2015,220:13-17.
[15]Zhang Y B,Wang Y J,Yang W R,et al.A rapid immunochromatographic test to detect the lily mottle virus[J].Journal of Virological Methods,2015,220:43-48.
[16]Lee J A,Choi S K,Yoon J Y,et al.Variation in the pathogenicity of lily isolates of cucumber mosaic virus[J].Plant Pathology Journal,2007,23:251-259.
[17]Liu Y H,Huang C J,Chen C Y.Evidence of induced systemic resistance against Botrytis elliptica in lily[J].Phytopathology,2008,98:830-836.
[18]Curir P,Guglieri L,Dolci M,et al.Fusaric acid production by Fusarium oxysporumf.sp lilii and its role in the lily basal rot disease[J].European Journal of Plant Pathology,2000,106:849-856.
[19]李谋强,陈君良,黄彦玮,等.不同海拔区兰州百合(Lilium davidii.var.unicolor)栽培群体遗传多样性[J].中国沙漠,2016,36(4):1029-1033.
[20]Hoegen E,Stromberg A,Pihlgren U,et al.Primary structure and tissue-specific expression of the pathogenesis-related protein PR-1bin potato[J].Molecular Plant Pathology,2002,3:329-345.
[21]Talley K,Alexov E.On the pH-optimum of activity and stability of proteins[J].Proteins,2010,78:2699-2706.
[22]Qing G L,Ma L C,Khorchid A,et al.Cold-shock induced high-yield protein production in Escherichia coli[J].Nature Biotechnology,2004,22:877-882.