Isolation of Trichoderma reesei pyrG Negative Mutant by UV Mutagenesis and Its Application in Transformation |
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Authors: | LONG Hao WANG Tian-hong ZHANG Ying-kuan State Key Laboratory of Microbial Technology Sh ong University Jinan P. R. China |
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Affiliation: | State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, P. R. China |
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Abstract: | Two uridine auxotrophic mutants of Trichoderma reesei were isolated by resistance to 5-fluoroorotic acid after UV mutagenesis.One mutant,called M23,was complemented with the Aspergillus niger pyrG gene carded by plasmid pAB4-1.A mutated pyrG gene of M23 was cloned and DNA sequencing analysis indicated that a cytosine was inserted into the 934-939 oligo dC position of the pyrG coding region,resulted in a frameshift mutation.Transformation efficiency was approximately 200-300 transformants per microgram of DNA with plasmid pAB4-1.Stable transformants were obtained by monosporic culture and showed to be prototroph after successive propagation.Vitreoscilla hemoglobin expression plasmid pUCVHb was cotransformed with plasmid pAB4-1 and attained a transformation efficiency of 71.8% or of 26.1% with pAN7-1.Southern blot analysis of the transformants demonstrated that plasmid pUCVHb was integrated into the chromosomal DNA.The experimental results demonstrated that the pyrG-based system was more efficient and timesaving than the conventional hygromycin B resistance-based transformation system. |
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Keywords: | Trichoderma reesei UV mutagenesis pyrG negative strain pyrG-based transformation system |
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