【作者】 王钰；

【导师】 李艳琴；

【作者基本信息】 山西大学 ， 生物化学与分子生物学， 2009， 硕士

【摘要】 生物防治是植物病害综合治理体系中的一个重要组成部分,植物同病原菌互作的进化过程中形成的过敏反应是自然界中植物抵抗病原菌侵染的最有效方式。Harpin是一类植物病原细菌分泌的蛋白质,能诱导非寄主植物产生过敏反应,这种防卫反应的产生,可以抵抗多种多样的病原物对植物的第二次侵染,类似于人或动物的免疫系统。枯草芽孢杆菌本身无毒,对多种植物病原菌具有拮抗作用,是一种天然的植病生防菌。利用Harpin的诱导抗病性和生防菌的拮抗作用开发新型的生物农药具有重要的理论意义和应用价值。本实验将利用枯草芽孢杆菌来表达Harpin蛋白,期望获得一种既能诱导植物抗性又能与植物病原菌竞争、拮抗的多功能生防菌。为了能使Harpin蛋白分泌到培养基中,与植物细胞直接作用,诱导植物的防卫反应。首先,我们以枯草芽孢杆菌168总DNA为模板扩增组成型强启动子P43和分泌效率较高的nprB信号肽,通过重组PCR连接两片段,克隆至大肠杆菌和芽孢杆菌的穿梭载体pUBC19,构建组成型表达分泌载体pUBC-PS。其次,以新鲜制备的胡萝卜软腐欧文氏杆菌(Erwinia carotovora subsp. carotovora)Se9总DNA为模板,设计引物扩增hrpNECC9基因,PCR产物克隆于表达载体pUBC-PS,获得重组质粒pUBC-PSh2,将重组质粒转入大肠杆菌DH5α,在含有100μg/mL氨苄青霉素的LB平板上筛选转化子-DH5a(pUBC-PSh2)。小量提取pUBC-PSh2质粒,采用碱金属转化法转入缺失8种蛋白酶基因的枯草杆菌WB800,在含有卡那霉素20μg/mL的LB平板上筛选工程菌,对其酶切、PCR验证结果表明：生防工程菌WB800(pUBC-PSh2)构建成功。SDS-PAGE分析表明：工程菌培养到72 h时,表达产物Harpin蛋白被分泌到细胞培养液中,表达量约占菌体蛋白总量的15%。由此证明,工程菌中的hrpNECC9基因在枯草杆菌P43启动子和nprB信号肽元件的带动下,不需诱导,实现了表达,并且,表达产物被分泌到胞外。生物活性检测和诱导植物抗病性分析结果表明：工程菌能够诱导烟草叶片产生过敏反应,并能提高番茄对早疫病菌的抗性。更多还原

【Abstract】 Biological control is an important component in plant diseases management system. Hypersensitive response is the most effective way in resistance pathogen infection. Harpin encoded by hrpN gene in E. carotovora subsp can induce hypersensitive reaction on non-host plants, once the establishments of systemic acquired resistance (SAR), plants exhibit a broad-spectrum of disease resistance against pathogen attack, similar to humans or animals immune system.Bacillus.subtilis is a natural plant bacterium with non-toxic, antagonism effect for plant pathogen. It would be feasible to explore biological control pesticides base on characteristics of Bacillus.subtilis and induction resistance of Harpin. This experiment will be used to express Harpin protein in Bacillus subtilis, in order to obtain multifunctional biocontrol bacteria that can induce plant resistance and compete with plant pathogen.In this research, firstly, by PCR using B.Subtilis168 genomic DNA as template, the P43 promoter (constitutive-expression) and the nprB signal peptide were amplified and connected with E.coli-B.subtilis shutter vector pUBC19, then formed expression vector pUBC-PS.Secondly,hrpNEcc9 from Erwinia carotovor (Se9) was cloned into vector pUBC-PS to obtain expression and secretion Harpin plasmid pUBC-PSh2, which was transformed into B. subtilis WB800 by alkali metals method. Next, the recombinant strain was cultured at 37℃, shaking 200 rpm for 72h. Then, the target protein was obviously detected in culture supernatant fluid by SDS-PAGE. And the protein expression quantity was about 15% of cell protein. At the same, Cell supernatant was injected into tobacco leaf which induced hypersensitive response. The induction of plant disease resistance analysis showed that:the engineering bacteria can improve resistance for Alternaria solani.更多还原