Construction And Expression Of Three CBH Gene Mutant Strains Of Neurospora Crassa

Cellulose is a kind of bio-energy which is widely distributed in nature.It's complex molecularstructure makes difficult to be broken down naturally.It is an effective and environmentally friendly way to solve the waste cellulose through microbial cellulase which is secreted by bacteria or fungi.In the process of cellulose degradation,3 types of cellulase are widely recognized exoglucanasea(CBH),endoglucanase(EG)and beta glucosidase(BG).Cellulase plays a very important role in Neurospora crassa.In this article we focus on 3 CBH genes they play the important roles in cellulose degradation of Neurospora crassa,setting out to construct 3 CBH gene deletion and CBHs over-expressed strains.The method of spore hybridization was used to build N.crassa'sdeletion mutation strains.On the basis of cbh-1(NCU07340),cbh-2(NCU09680),gh6-3(NCU07190)3single gene deletion mutation strains,finally were designed according to the Neurospora crassa Database.The PCR primers for 3 CBHs,and then the PCR products were connected to qa-5myc.6His carrier.The 3 double gene deletion strains and 1 triple gene deletion strains were constructed successfully.The 3 CBH genes over-expressed strains were screened out by Histidine label and Western hybrid examined that the over-expressed proteins of were 90 kda,70kda and 65 kda respectively.The results of comparison on growth phenotype between gene deletion and gene over-expressed strains discovered that cbh-2 gene missing could make hyph a growslowly,and the chb-2 single gene deletion strains,and the cbh-2+cbh-1 double gene strains and the cbh-2+gh6-3 double gene strains and the three gene deletion strainswere foundedthinnerhyphae and whit more branchescompared with wild type strains.Their spore morphology also changed after CBHs deletion or over-expressed.Whichmeant that CBH genes had an effect on the growth and development of Neurospora crassa.The CBHs activity indicated that all of the 3 CBH genes that CBH enzyme activity played an role on after deletion of cbh-2,cbh-1 or gh6-3,CBH enzyme activity reduced about 59%.CBH enzyme activity performed similar by deletion of cbh-2.The cbh-1 gene deletion made the CBH enzyme activity reduced about 48%.The gh6-3 gene deletionstrain was about 22%.What's intere sting was the enzyme activity of deletion strain of cbh-1+gh6-3 was higher than that of only cbh-1 or gh6-3 missing.An more complex system of cellulase components and expression,might be able to explain this,but further research on the transcription level are necessary.Measurment on cellulase activity of ?-glycosidase and endo show that the delection of genes result in decrease of endo-1,4-?-D-glucanase but ?-1,4-glucosidase remand unchangeable,Over-expression of CBHs had near no effect on both endo-1,4-?-D-glucanase but ?-1,4-glucosidase.Several over-expression strain CBH different carbon sources in different media activity,natural cellulose enzyme inducing better,compared to the wild type strain expressing CBH activity over several different degree.