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【目的】构建增强抑制真菌能力兼杀虫的苏云金芽胞杆菌多功能生防菌株。【方法】将含有组成型高效表达启动子、地衣芽胞杆菌chi MY基因的重组质粒p DM,转化进杀虫活性高且有一定抑菌活性的Bt519-1菌株。酶谱分析方法确认Bt519(p DM)组成型异源表达几丁质酶。室内测定工程菌株抑菌谱,计算抑菌效率,确定最敏感的植物病原真菌,进行植物盆栽病害防治的应用潜力评价。将不同浓度的Bt粗酶液灌入甜椒幼苗根部,12 h后接种辣椒疫霉孢子液,接种2 d后开始观察,记录发病株数。自7 d起调查植株发病情况统计并分析防治效果。【结果】SDS-PAGE及酶谱分析证明,Bt519(p DM)能够特异表达68 k D蛋白,该蛋白为异源几丁质酶Chi MY。抑菌谱测定证明,工程菌抑制效率达到90%以上的有5种真菌,其中最明显的是辣椒疫霉。盆栽实验证明,Bt519(p DM)7 d的防效为73.2%。工程菌株对棉铃虫的半致死浓度(LC50)为121.26 mg/L。【结论】Bt519(p DM)是一株有应用潜力的生防菌株。
【Objective】 To construct a multi-functional biocontrol strain of Bacillus thuringiensis that enhances the ability of inhibiting fungi and killing insects. 【Method】 The recombinant plasmid p DM containing the constitutively highly expressed promoter and the chi MY gene of Bacillus licheniformis was transformed into Bt519-1 strain with high insecticidal activity and antibacterial activity. Zymogram analysis confirmed the constitutive heterologous expression of chitinase by Bt519 (p DM). Indoor determination of antibacterial spectrum of engineering strains, calculation of antibacterial efficiency, to determine the most sensitive plant pathogenic fungi, plant pot disease prevention and control of potential applications. Different concentrations of Bt crude enzyme solution were poured into the roots of sweet pepper seedlings and inoculated with Phytophthora capsici spores after 12 h. The number of infected plants was observed after 2 d of inoculation. From 7 d investigation of plant disease incidence statistics and analysis of control effect. 【Result】 SDS-PAGE and zymography showed that Bt519 (p DM) could specifically express 68 kD protein, which is a heterologous chitinase Chi MY. According to the antibacterial spectrum test, five kinds of fungi were found to inhibit the efficiency of engineering bacteria to over 90%. The most obvious one was Phytophthora capsici. Pot experiment showed that the control effect of Bt519 (p DM) for 7 days was 73.2%. The LC50 of the engineered strains against cotton bollworm was 121.26 mg / L. 【Conclusion】 Bt519 (p DM) is a biocontrol strain with potential application.