DOI: 10.3724/SP.J.1145.2013.00986

Chinese Journal of Appplied Environmental Biology (应用与环境生物学报) 2013/19:6 PP.986-989

Cloning and High Expression of HarpinEa USAW004 Gene of Erwinia amylovora USAW004 Strain

From apple shoots infected with fi re blight, we separated and puri fi ed Erwinia amylovora strain USAW004, which caused hypersensitive responses (HR) in non-host plant tobacco leaves. By constructing a USAW004 genomic library and in situ colony hybridization, HarpinEaUSAW004 gene positive clone was screened, and the corresponding fragment was subcloned into the vector pBluescript-II SK(+). An orf of 1 212 bp of HarpinEaUSAW004 gene was determined in the positive clone, with the transcription product HarpinEaUSAW004 consisted of 403 amino acid residues. By cloning HarpinEaUSAW004 gene to the expression vector pET28a(+) under control of the T7 promoter, we successfully constructed an expression plasmid pT7-HrpEaUSAW004. The product HarpinEaUSAW004 was induced by IPTG which expressed in E.coli JM109 with a high effi ciency, also inducing a typical HR in tobacco leaves. Fig 6, Ref 10

Key words:Erwinia amylovora,clone,HarpinEaUSAW004 gene,HarpinEaUSAW004 protein,plant hypersensitive response

ReleaseDate:2015-04-15 08:42:21

[1] Dong HS, Delaney TP, Bauer DW, Beer SV. Harpin induces diseaseresistance in Arabidopsis through the systemic acquired resistancepathway mediated by salicylic acid and the NIM1 gene [J]. Plant J, 1999, 20: 107-215

[2] Lehtimaki S, Rantakari A, Routtu J, Tuikkala A, Li J, Virtaharju O, Palva ET, Romantschuk M, Saarilahti HT. Characterization of the hrppathogenicity cluster of Erwinia carotovora subsp. carotovora: highbasal level expression in a mutant is associated with reduced virulence [J].Mol Genet Genomics, 2003, 270: 263-272

[3] Van der Zwet T, Keil HL. Importance of pear-tissue injury to infection byErwinia amylovora and control with streptomycin [J]. Can J Microbiol, 1979, 18: 893-900

[4] Wei ZM, Laby R, Zumoff C, Bauer D, He S, Collmer A, Beer S. Harpin, elicitor of the hypersensitive response produced by the plant pathogenErwinia amylovora [J]. Science, 1992, 257: 85-88

[5] 任欣 正 . 植物病原细菌的分类和鉴定 [M]. 北京: 中国农 业出版社, 1994 [Ren XZ. Classification and Identification of Plant PathogenicBacteria [M]. Beijing: China Agriculture Press, 1994]

[6] 方中达. 植病研究方法[M]. 3版. 北京: 中国农业出版社, 1998 [FangZD. Plant Pathology Research Methods [M]. 3rd ed. Beijing: ChinaAgriculture Press, 1998]

[7] Sambrook J, Russell DW. Molecular Cloning Experiments Directory [M].Beijing: Science Press, 2005

[8] Bauer DW, Wei ZM, Beer SV, Collmer A. Erwinia chrysanthemiharpinEch: an elicitor of the hypersensitive response that contributes tosoft-rot pathogenesis [M]. Mol Plant-Microb Interact, 1995, 8: 484-491

[9] 董宏平, 李明立, 彭建令, 于海芹, 董汉松. HarpinEa促进烟草和蕃茄生长和诱导抗虫的信号通路[J]. 高技术通讯, 2004, 12: 29-32 [DongHP, Li ML, Peng JL, Yu HQ, Dong HS. Growth and insect resistancepathways activated by HarpinEa in tobaco and tomato [J]. High-techCommun, 2004, 12: 29-32]

[10] 程茂高, 乔卿梅, 蒋士君. Harpin Ea激 发烟草叶片过敏性 反应与苯丙氨酸解氨酶活性的关系 [J]. 烟草科 技, 2007, 11: 63-66 [ChengMG, Qiao QM, Jiang SJ. Relationship between anaphylaxis elicited byHarpinEa and phenylalanine ammonialyase activities in tobacco leaves[J]. Tobaco Sci Technol, 2007, 11: 63-66]