首页> 外文期刊>Molecular biology reports >Towards a novel efficient T-DNA-based mutagenesis and screening system using green fluorescent protein as a vital reporter in the industrially important fungus Trichoderma reesei.
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Towards a novel efficient T-DNA-based mutagenesis and screening system using green fluorescent protein as a vital reporter in the industrially important fungus Trichoderma reesei.

机译:迈向新型高效的基于T-DNA的诱变和筛选系统,该技术使用绿色荧光蛋白作为工业重要真菌里氏木霉中的重要报告物。

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Agrobacterium-mediated T-DNA transfer has been proven to be an efficient strategy for insertional mutagenesis and elucidation of gene function in filamentous fungi. The implementation of large-scale T-DNA insertional mutagenesis requires the development of high-efficient transformation and high-throughput screening procedures. Here, using green fluorescent protein (GFP) as a vital marker, a highly efficient T-DNA-based mutagenesis and screening system was developed in Trichoderma reesei. The uridine auxotrophic T. reesei M23 as the host was transformed with A. tumefaciens EH105 strain harboring a binary vector pC-OEP, which beared the pyrG gene for primary selection on minimal medium without uridine and the egfp gene for fluorescence-based rapid screening of the mitotically stable transformants. The efficiency of transformation was up to 10-20 transformants per 10(5) target conidia. Microscopic examination revealed strong GFP expression and fluorescence emission in conidia, growing hyphae and mycelia. An effective and convenient screening procedure using 96-well plates and multilabel counter for fluorescence intensity counting was developed to rapidly identify the T-DNA tagged T. reesei mutants. Furthermore, the presence of T-DNA integration at random sites in the genome was confirmed by Southern blot analysis. This report of the T-DNA-based mutagenesis and rapid screening system using GFP as a vital reporter provides a promising strategy to speeding up the genome-scale T-DNA insertional mutagenesis and functional genomics analysis of this cellulolytic fungus T. reesei.
机译:农杆菌介导的T-DNA转移已被证明是一种有效的策略,用于丝状真菌的插入诱变和基因功能的阐明。大规模T-DNA插入诱变的实施需要开发高效转化和高通量筛选程序。在这里,使用绿色荧光蛋白(GFP)作为重要标记,在里氏木霉中开发了一种高效的基于T-DNA的诱变和筛选系统。用带有二元载体pC-OEP的根癌农杆菌EH105菌株转化尿嘧啶营养缺陷型里氏木霉M23作为宿主,该菌株携带pyrG基因用于在没有尿苷的基本培养基上进行初步选择,并带有egfp基因用于基于荧光的快速筛选有丝分裂稳定的转化子。每10(5)个目标分生孢子转化效率最高为10-20个转化子。显微镜检查显示在分生孢子,生长的菌丝和菌丝体中强烈的GFP表达和荧光发射。开发了一种有效且方便的筛选程序,使用96孔板和多标记计数器进行荧光强度计数,以快速识别T-DNA标签的里氏木霉突变体。此外,通过Southern印迹分析证实了基因组中随机位点处T-DNA整合的存在。这份基于GFP的T-DNA诱变和快速筛选系统的报告为重要的报告者,提供了一种有前途的策略,可加快这种纤维素分解真菌里氏木霉的基因组规模T-DNA插入诱变和功能基因组学分析。

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