Gene Clone And Bioinformatics Analysis Of Trichoderma Reesei Exoglucanase1

Trichoderma reesei cbh1was identified as the main study object by analyzing the results for the activity determination of exoglucanase1, endoglucanase andglucosidase, target gene sequences containing promoter and terminator were amplified successfully by PCR, connected to pUC118Vector by Blunt-end ligation, BLAST analysis was carried on after cloned and sequenced, these layed the foundation for further research of exoglucanase1. The main results were illustrated as follows:1Trichoderma reseei had the highest enzyme activity at the sixth day, highest exoglucanase activity was160.921U/mL, highest endonuclease activity was125.297U/mL, the highest beta-glycosidase activity was45.525U/mL, the highest filter enzyme activity was124.715U/mL; The highest exoglucanase activity as four times as β-glycosidase activity. Trichoderma reseei showed a high level of exoglucanase activity.2In PCR amplification of the gene exoglucanase1, the optimal DNA template was1.0μL, the optimum dNTP was1.3μL, The best annealing temperature was67.5℃.3With the proportion of1to4for the pUC118vector and exoglucanase1, the connection is more efficient. The effect of transformation into E. coli JM109was good at16℃for the night.4.Sequencing result suggested that the target gene fragment with both promoter and terminator have been successfully amplified. Amplified DNA sequence was3496bp in length, the upstream promoter sequence was1500bp, the downstream terminator sequence was600bp, compared with the registered GL985084Trichoderma reesei exoglucanase1, there was99%homology. There were3exons and2introns in the DNA sequence. The first exon encoded signal peptide, the next two exons encoded mature peptide. Open reading frame consists of514amino acids, speculated that the molecular weight was57kDa.5. Through bioinformatics analysis Trichorderma reseei cbhl has3CDS:459bp,(1499-1959) encodes153aa,697bp(2028-2724) encodes232aa,276bp(2899-3174) encodes92aa; Initiation codon is ATG(1499-1501), Termination codon is TAA(3172-3174). Cbh1makes its core promoter TATA frame (GTATATATA) at1366bp, It is both transcriptional binding sites and transcription initiation site.