Towards a novel efficient T-DNA-based mutagenesis and screening system using green fluorescent protein as a vital reporter in the industrially important fungus <Emphasis Type="Italic">Trichoderma reesei</Emphasis> |
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Authors: | Yaohua Zhong Haina Yu Xiaoli Wang Yi Lu Tianhong Wang |
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Institution: | (1) State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, 27 Shanda South Road, Jinan, 250100, People’s Republic of China; |
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Abstract: | 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 105 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. |
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Keywords: | |
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