Vector Construction And Expression Of The Neurospora Crassa Exoglucanase Gene

Plant cellulose is the most abundant renewable carbon resources on earth,but it is difficult to be decomposed because of its linear giant molecular structure.Using the method of cellulase hydrolyzing is an effective and environmental solution to utilize the waste cellulose.Neurospora crassa is an important model microorganism that can produce cellulase.In this article,we mainly focused on cellulases genes from Neurospora crassa,and then successfully constructed a strain modified with high-expressed cellulase.First,we selected 10 mutants from the mutant library of Neurospora crassa which gene suspected to be relevant with cellulase.By enzyme activity measurement of three kinds of cellulase,exoglucanase,endoglucanase and ?-glucosidase,the exoglucanase activities of ?NCU09680 was significantly decreased,also the mycelial growth and the degree of spore germination showed lower level compared to wild-type strain.Therefore we considered that the protein encoded by this gene was related to cellulase activity.Next we cloned NCU09680 gene according to its gene sequence reported from Neurospora Crassa Database,and then connected to a vector of qa-2.5 myc.6HisdH,the newly constructed plasmid is 8759bp in length,and has a qa promoter sequence,an Amp sequence,his3 sequence and a 6His tag.Aiming at high-expressed cellulase,vector of qa-2.5 myc.6HisdH-NCU09680 was transformed into a histidine-deficient strain 301-6 of Neurospora crassa by electroporation method.The western blotting was used to detect the expression of exoglucanase protein,totally 10 transformed strains were obtained.The result of SDS-PAGE electrophoresis from fermentation broth of transformed strains showed the protein size was 62kDa,which could be effiently secreted out of the cell,and the protein amount expressed was larger than the wild-type strain WT87-3 and ?NCU09680,this indicated that the expression of exoglucanase from high-expressed strains was improved.By cultured to the tenth generation,the exoglucanase strains still had high cellulase activity.