Improving cellulase expression of Trichoderma reesei by RNAi- mediated repression of cre1 transcription

The RNA interference( RNAi) technique was used to repress the expression of the main transcriptional repressor cre1 in order to improve the cellulase production of Trichoderma reesei. The cbh1 promoter,a reversed cre1 gene fragment( 568 ~ 963 bp),an ordinary cre1 gene fragment( 655 ~ 961 bp),and cbh2 terminator were all amplified from the genomic DNA of T.reesei by polymerase chain reaction( PCR).The DNA assembler method was used to assemble all these gene fragments in pRS424 to obtain the p Cre1-i plasmid.Two fragments encompassing the RNAi cassette were amplified from the plasmid and transformed simultaneously into T.reesei.PCR was used to verify the positive colonies.In the flask batch culture,one of the transformants displayed 0.67、3.70 and 0.46 U/m L for the filter paper cellulase,endoglucanase,and CBHI activity,which were 1.3,1.8,and 5.6 folds of the parent strain.By using RT-q PCR,the transcript level of cre1 in this transformant was determined to be lowered to 43% of that of the parent strain.