Isolation of Trichoderma reesei PyrG negative mutant by UV Mutagenesis and its Application in Transformation

Long Hao; Wang Tianhong; Zhang Yingkuan

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Isolation of Trichoderma reesei PyrG negative mutant by UV Mutagenesis and its Application in Transformation

Long Hao,Wang Tianhong * #,Zhang Yingkuan

State Key Laboratory of Microbial Technology, Shandong University

*Correspondence author

#Submitted by

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Funding: 国家自然科学基金,教育部博士点基金等（No.30470052,20040422042）

Opened online:17 December 2007

Accepted by: none

Citation: Long Hao,Wang Tianhong,Zhang Yingkuan.Isolation of Trichoderma reesei PyrG negative mutant by UV Mutagenesis and its Application in Transformation[OL]. [17 December 2007] http://en.paper.edu.cn/en_releasepaper/content/17054

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 carried by plasmid pAB4-1. Mutated pyrG gene of M23 was cloned and DNA sequencing analysis indicated that a cytosine inserted into the 934-939 oligo dC position of the pyrG coding region, resulting in a frameshift mutation. Transformation efficiency was approximately 200-300 transformants per μg 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 co-transformed with plasmid pAB4-1 attained transformation efficiency of 71.8% versus 21.6% 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.

Keywords:Trichoderma reesei, UV mutagenesis, pyrG negative strain, pyrG-based transformation system

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