表达Harpin蛋白的芽孢杆菌工程菌的构建及其生防效果  

Construction of Harpin expression engineering strain FZBHarpin and evaluation of its biocontrol activity

作　　者：乔俊卿[1,2] 伍辉军[1] 霍蓉[1] Rainer Borriss 高学文[1] 

机构地区：[1]南京农业大学植物保护学院/农作物生物灾害综合治理教育部重点实验室,江苏南京210095 [2]江苏省农业科学院植物保护研究所,江苏南京210014 [3]德国柏林洪堡大学生物学和细菌遗传学实验室

出　　处：《南京农业大学学报》2013年第6期37-44,共8页Journal of Nanjing Agricultural University

基　　金：国家863计划项目(2012AA101504);国家公益性行业(农业)科研专项(201303015);国家自然科学基金项目(31100056);高等学校博士学科点基金项目(20100097120011)

摘　　要：利用同源重组方法将来源于水稻细菌性条斑病菌的Harpin(HpaG Xooc)编码基因hpa1分别插入根围拮抗解淀粉芽孢杆菌FZB42的蛋白水解酶编码基因aprE和nprE位点,成功构建双拷贝Harpin表达工程菌株FZBHarpin。反转录PCR检测结果显示,hpa1能在RNA水平上正常转录表达。工程菌FZBHarpin发酵72 h后,其发酵液可引起烟草过敏性反应,这表明Harpin蛋白外泌胞外并保持生物活性。利用FZBHarpin浸种烟草后,烟草主根长达64.05 mm,比对照增加了30%,促生效果是FZB42的2倍。盆栽水稻白叶枯病防治试验结果显示,FZBHarpin防效为51.9%,比出发菌株FZB42防效显著提高。这为利用病原物中可激发植物产生抗病性的激发子来遗传改良生防微生物提供了理论基础。HpaGXcoc from rice pathogenic bacterium Xanthomonas oryzae pv. oryzicola, is a member of the Harpin group of proteins, eliciting hypersensitive cell death in non-host plants,inducing disease and insect resistance in plants and enhancing the plant growth. Bacillus amyloliquefaciens FZIM2 has been used as powerful weapons for the suppression of plant pathogenic organisms and plant growth promotion in the world. In this study,two recombinant Harpin protein expression vectors pGAprEHS and pUNprEKHS were con- structed and integrated into the aprE,nprE site of FZB42. These two vectors were composed of powerful promoter P43 and nprB signal peptide,which leads to the expression of Harpin protein continuously and secretes into the medium. The result of hypersensitive response（HR） on tobacco also indicates that Harpin protein is secreted in the extracellular. RT-PCR analysis reveals the hpal gene can be transcribed normally. After tobacco seeds soaking by FZB derived strains, FZBHarpin exhibits an excellent effect of plant growth pro- motion;the tobacco root length is about 64.05 mm and is 30% higher than control. The greenhouse experiment revealed that engineered strain FZBHarpin is the best one for the control of rice bacterial leaf blight; the control efficacy is 51.9%. This study provides a fundamental basis for genetically modification of biocontrol agent by using of HR-elicitors from plant pathogens.

关 键 词：解淀粉芽孢杆菌FZB42 HARPIN蛋白 基因工程菌 生物防治 

分 类 号：S476.8[农业科学—农业昆虫与害虫防治]