农杆菌介导的获取疏绵状嗜热丝孢菌插入突变体体系的建立

Agrobacterium tumefaciens-mediated transformation as a tool for insertional mutagenesis in thermophilic fungus Thermomyces lanuginosus

摘要:

摘要:【目的】建立疏绵状嗜热丝孢菌的稳定遗传转化体系并获得插入突变体。【方法】利用农杆菌介导的方法建立疏绵状嗜热丝孢菌的遗传转化体系;分别通过Southern杂交、克隆转移DNA(T-DNA)侧翼序列来确定T-DNA 在疏绵状嗜热丝孢菌基因组中的拷贝数和插入位点。【结果】成功建立了可靠的疏绵状嗜热丝孢菌的遗传转化体系。共培养过程中使用萌发孢子是成功建立疏绵状嗜热丝孢菌遗传转化体系的必要条件。疏绵状嗜热丝孢菌萌发的孢子与农杆菌在28℃共培养48h时，转化效率最高。乙酰丁香酮(AS)在农杆菌预培养及疏绵状嗜热丝孢菌萌发的孢子与农杆菌的共培养阶段都是必需的，且在共培养阶段当AS浓度为500 μM时转化效率最高。Southern 杂交验证表明，79.2%的转化子为T-DNA单拷贝插入，且通过热不对称PCR(TAIL-PCR)分析得出T-DNA在该菌基因组中的插入位点是随机的。通过该转化系统筛选到部分表型突变体。【结论】我们首次报道了利用ATMT技术成功转化嗜热真菌-疏绵状嗜热丝孢菌，证明了该方法是一种简单有效的获得插入突变体的方法，并为该嗜热真菌进行基因定位提供了工具。

Abstract:

Abstract:［Objective］To establish a stable transformation system of the thermophilic fungus Thermomyces lanuginosus for its insertional mutagenesis.［Methods］ Agrobacterium tumefaciens-mediated transformation (ATMT) was applied to establish transformation system of T.lanuginosus. Southern blotting of hph gene and cloning of transforming DNA (TDNA) flanking sequences were used to determine insert number and site of T-DNA in the fungal genome，respectively．［Results］A reliable transformation method is established for T．lanuginosus．Specifically，pre-germinating spores of T.lanuginosus used at co-cultivated period was a prerequisite．T.lanuginosus germinating spores co-cultivated with Agrobacterium tumefaciens at 28℃ for 48 h achieved the highest transformation efficiency．Addition of Acetosyringone (AS) during pre-culture of A．tumefaciens and co-cultivation of T．lanuginosus germinating spores with A．tumefaciens was essentially required，and the best results were obtained with AS at the concentration of 500μM．Southern blotting analysis showed that majority of transformants (79.2%) contained a single insertion of T-DNA.Thermal asymmetric interlaced PCR (TAIL-PCR) analysis showed random insertion of T-DNA in the fungal genome．Using the transformation system，some stable phenotypic mutants of T．lanuginosus were obtained．［Conclusion］We report，for the first time，a simple and efficient method for transforming T. lanuginosus by using ATMT．This approach provides a tool for insertional mutagenesis gene tagging in this thermophilic fungus.