转异源chi基因的Bt工程菌株的生防潜力评价

Biocontrol potential of Bacillus thuringiensis strain Bt519-1 transformed with heterologous chitinase gene

摘要:

【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chiMY基因的重组质粒pDM，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(pDM)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(pDM)能够特异表达68 kD蛋白，该蛋白为异源几丁质酶ChiMY。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(pDM) 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(pDM)是一株有应用潜力的生防菌株。

Abstract:

[Objective] To produce Bacillus thuringiensis Bt519-1 as biocontrol agent with enhanced antifungal and insecticidal activity. [Methods] A recombinant plasmid, pDM, containing a constitutive high expression promoter and the chitinase gene chiMY from B. licheniformis, was constructed and transferred into strain Bt519-1 by electroporation. Zymogram analysis was used to test the constitutive expression of chitinase of strain Bt519(pDM). Inhibitory activity of the engineered strain was measured against 10 species of fungi that are plant pathogens. The most sensitive fungus was selected to test in pot culture experiments. Roots of Capsicum annuum seedlings were irrigated with various concentrations of the Bt crude enzymes, inoculated with the fungal sporangial suspension 12 h later. The plant disease index and control efficacy were determined 7 d after inoculation. [Results] SDS-PAGE and zymogram analysis confirmed that strain Bt519(pDM) expressed chitinase (68 kD). Inhibitory of Bt519(pDM) was effective for 5 of the 10 tested fungi of which Phytophthora capsici was most sensitive. Pot culture experiments demonstrated that the control efficacy of Bt519(pDM) against P. capsici was 73.2%. The half lethal concentration (LC50) of Bt519(pDM) against the moth Heliothis armigera was 121.26 mg/L. [Conclusion] Bt519(pDM) has potential as an antifungal and insecticide.