植物内生生防蜡样芽孢杆菌0-9菌株转座子插入突变体库的建立及评价的开题报告.docxVIP

植物内生生防蜡样芽孢杆菌0-9菌株转座子插入突变体库的建立及评价的开题报告 Title: Establishment and Evaluation of a Transposon Insertion Mutant Library of Endophytic Biocontrol Bacillus thuringiensis Strains 0-9 Introduction: Endophytic biocontrol Bacillus thuringiensis (Bt) strains have been widely investigated as a promising alternative for chemical pesticides because of its specificity, eco-friendliness, and high efficiency. However, limited genetic information and the complexity of endophytic Bt strains colonization in the plant host hamper the identification and characterization of functional genes responsible for biological control. Transposon insertion mutagenesis provides an efficient technique to randomly inactivate genes and identify genes associated with a specific phenotype. In this study, we aim to establish a transposon insertion mutant library for Bt strains 0-9 isolated from apple trees and evaluate their biocontrol efficacy against apple tree pathogens. Methods: 1. Bt strains 0-9 were grown in Luria-Bertani medium at 28°C. 2. The transposable element Tn5 was used to generate a transposon library according to the manufacturers protocol. 3. Colonies with transposon insertion were selected by kanamycin resistance and confirmed by PCR using Tn5-specific primers. 4. Approximately 10,000 insertion mutants were pooled and stored at -80°C as a mutant library. 5. To evaluate the biocontrol efficacy of the mutant library, apple trees were inoculated with pathogens, including Erwinia amylovora, Alternaria alternata, and Botryosphaeria dothidea, in a greenhouse. 6. The plant tissues were collected after 7 days, and bacterial colony-forming units (CFUs) were counted. 7. The biocontrol efficacy of the mutants was evaluated by comparing the bacterial CFUs of the mutants and the wild-type strains. Expected Results: The establishment of a transposon insertion mutant library for endophytic biocontrol Bt strains 0-9 will provide a powerful tool for identifying functional genes responsible for biological