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 p...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. 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.展开更多
基金the National Natural Science Foundation of China(No.30470052)the National Basic Research Program of China(Nos.2003CB716006 and 2004CB719702)the Natural Science Research Foundation for the Doctoral Program of Edu-cation Ministry of China(No.20040422042).
文摘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. 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.
