里氏木霉RL-P37尿嘧啶营养缺陷型菌株的构建

里氏木霉（Trichoderma reesei） RL-P37作为纤维素酶高产菌,被广泛地应用在工业蛋白的生产过程.从基因水平对工业菌株进行分子改造已经成为提升工业菌株生产性状的重要手段.因此,亟需构建适用于基因敲除以及外源蛋白表达的营养缺陷型菌株,特别是可用于基因无痕敲除的尿嘧啶营养缺陷型菌株.本研究以潮霉素为标记,成功构建了里氏木霉RL-P37尿嘧啶营养缺陷型菌株,并得到Southern验证.该菌株在含有1.5 mg/mL 5-氟乳清酸、2 mg/mL尿苷的MM培养基中可以正常生长,并且在不含有尿苷的MM培养基上不能生长.此外,利用粗糙脉孢菌（Neurospora crassa）的pyr4基因对该突变体进行了回补.里氏木霉RL-P37尿嘧啶营养缺陷型菌株的构建为该菌纤维素酶表达和分泌的机理研究以及后续产酶性能分子改造奠定了物质基础.

Construction of uracil-auxotrophic strain of Trichoderma reesei RL-P37

As a cellulase high-yielding strain,Trichoderma reesei RL-P37 has been widely used in industrial protein production process.It is an important method that the properties of industrial strains could be improved by genetic manipulation.So the uracil-auxotrophic strain suitable for gene knockout and heterologous protein expression needed to be constructed.This system could be employed to introduce or delete desired genes without leaving any undesired DNA.In this paper,hygromycin was used as a selective marker,uracil-auxotrophic T.reesei RL-P37 strain was successfully constructed and the result was verified by Southern blotting.The mutant could grow well in MM containing 1.5 mg/ml 5-FOA and 2 mg/ml uridine,but could not grow on MM medium without uridine.In addition,this mutant can restore to prototrophy with pyr4 gene from Neurospora crassa.The establishenmen of uracil-auxotrophic strain of T.reesei RL-P37 could be used as a tool for improvement of the productivity of the industrial strains and therefore facilitated the study of the mechanism of cellulase expression and secretion.