球孢白僵菌T-DNA突变体库的构建及高温和高渗敏感型突变体的筛选

Construction of Beauveria bassiana T-DNA insertion mutant collections and identification of thermosensitive and osmosensitive mutants

摘要:

摘要：【目的】筛选对高温和高渗等逆境胁迫敏感的球孢白僵菌T-DNA随机插入突变体，并克隆相关基因，为研究杀虫真菌适应逆境的分子机理奠定基础。【方法】利用逆境胁迫的方法从球孢白僵菌的T-DNA随机插入突变库中筛选对高温和高渗敏感的突变体，进而利用YADE (Y-shaped adaptor dependent extension)技术克隆相关基因。【结果】筛选得到5个对高温和高渗敏感的突变体。其中2个（212和2550）对高温敏感，在32oC下生长完全受抑；其它3个突变体对高渗胁迫敏感。突变体212的分生孢

Abstract:

Abstract: [Objective] This study was to identify genes involved in thermotolerance and osmotolerance of Beauveria bassiana. [Methods] A collection of T-DNA random insertion mutagenesis was constructed and thermosensitive and osmosensitive mutants were screened. The DNA fragments flanking to inserted T-DNA sequence in the mutants were isolated by using Y-shaped adaptor dependent extension (YADE) method. [Results] Five mutants impaired in thermotolerance or osmotolerance were identified and characterized. Mutant 212 and 2550 could not grow at 32oC, while mutants 139, 2737 and 2812 showed depressed growth under osmotic stress. Conidial viabilities of 212 was decreased by 23%, 52% and 21% after stressed by ultraviolet, heat shock, and H2O2 oxidation, respectively, compared to the wild-type strain. Virulence of mutant 212 and 2737 against aphids Myzus persicae were reduced by 12% and 40%, respectively. Sequencing analysis indicated that two function-unknown genes were interrupted by insertion of T-DNA in mutant 212 and 2737. In other three mutants, the insertions are located in the non-encoding regions. [Conclusion] The results demonstrate the successful use of T-DNA insertion mutagenesis in identification of genes involved in thermotolerance and osmotolerance of B. bassiana.