Cloning Of Cellulase Genes From Trichoderma Longibrachiatum And Construction Of Heterogeneous Gene Expression System Of Trichoderma Reesei

In recent years, fuel ethanol as a cleaner energy with excellent performance hasattracted great attention while the reserves of traditional fossil fuels are being drasticallyreduced by over-exploitation. Lignocellulose is the most abundant biomass in the earth, itwill displays great economic and social significance in improving the current scarcity ofenergy if construction of high-yield cellulase strains for efficient hydrolysis of celluloseinto glucose and fermentation for the production of fuel ethanol. Trichoderma reesei is oneof the effective cellulose-degrading microorganisms, so construction of Trichoderma reeseistrain with high-yield cellulase is an important means for optimizing the cellulosecomponent and improving the abilities of cellulose degradation.Seven strains producing cellulase were isolated from cellulose-rich environment andcultured in liquid medium for testing the abilities of producing cellulase. The resultsshowed that strain FU05produced cellulase with maximal activity, which reached106.4U/mL. Based on the cell morphology and analysis of some biochemical and physiologicalcharacteristics, strains FU01, FU02, FU03and FU04were identified as Penicillium whilestrains FU05, FU06, FU07were identified as Trichoderma. Furthermore, the latter threestrains were identified as Trichoderma longibrachiatum through ITS sequence analysis.Accordingly, the cellulase genes of bgl2, cbh2and eg1were obtained fromTrichoderma longibrachiatum FU05, FU06, FU07by PCR. Sequence analysis showed thatthe homology of bgl2gene among strains FU05, FU06and FU07reached99.49%, withonly22-base difference in gene sequence. At the same time, the data also displayed thehomology of cbh2gene among these three strains mentioned above reached99.45%and thehomology of eg1gene reached99.11%. By conducting sequence alignment analysis withthe cellulase genes in GenBank, data showed that the homology was over90%betweenstrain FU05and other Trichoderma strains. Consequently, the corresponding amino acidsequences were also quite similar. By means of PROSITE motif search, the locations ofN-glycosylation site, cellulose-binding domain and conserved domains of glycosylhydrolases family in the corresponding protein were confirmed.To construct the heterogeneous gene expression vector of Trichoderma reesei, firstlythe Trichoderma reesei strong cbh1promoter and terminator were inserted into the plasmid pUC19, which was used as a backbone. Then the vector pUC19-PTM was obtained byinserting multiple cloning sites into the place between the promoter and terminator.Subsequently, the vector pUC19-PTMG was constructed by inserting the kanMX gene as aselection marker into multiple cloning sites. If the kanMX gene was replaced with bgl2gene,the vector pUC19-PTMB would be achieved. When the vector was introduced intoprotoplasts of Trichoderma reesei along with the vector pUC19-PTMG which has theselection marker mentioned above, the transformants grown on the selective mediumcontaining G418would be expected. However, the transformants did not appear on theplate for some unknown reasons.At present, few papers about the cellulase genes of Trichoderma longibrachiatum havebeen found. In our work, three cellulase-production strains of Trichodermalongibrachiatum had been screened and their cellulase genes were obtained by PCR, andthe cellulase genes were conducted to sequence alignment analysis, which formed a groundfor further studies on cellulase genes. Moreover, the construction of heterogeneous geneexpression vector of Trichoderma reesei for expression of bgl2gene from Trichodermalongibrachiatum FU05had been performed. Although there was no transformant selected,it still appeared not only as a significant attempt for the utilization of Trichoderma reesei asan expression host for building strong expression system, but also as a valuable referencefor future researches.