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(英文)

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

【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32℃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的分生孢子对逆境胁迫的抗性也显著降低。与野生菌株相比,分生孢子经紫外线、高温和氧胁迫后,其存活率分别下降了23%、52%和21%。突变体212和2737对桃蚜的致死率(接种浓度为1×107个孢子/mL)分别下降了12%和40%。T-DNA侧翼序列分析显示,突变体212和2737的2个功能未知基因的编码框被T-DNA随机插入阻断,而其它3个突变体的T-DNA插入位点位于非编码区域。【结论】本研究结果表明,利用T-DNA随机插入突变筛选高温与高渗等逆境相关突变体,进而克隆相关基因是一种行之有效的方法。

[Objective]This study was to identify genes involved in thermotolerance and osmotolerance ofBeauveria 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 growat 32℃, 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.