Study On Transcription Factors Of The Cellulose-Related Hydrolase In Trichoderma Orientalis EU7-22

Traditional fossil fuels can not meet the needs of human sustainable development.Looking for clean and economical renewable energy sources is one of the main problems in today's society.Cellulosic biomass mainly composed of cellulose and hemicellulose.Microbial hydrolytic enzymes,especially those from different species of fungi,play the main role in the degradation of lignocellulosic biomass.It is meaningful to investage the mechanism of enzyme production in filamentous fungi.Trichoderma orientalis EU7-22 was obtained by mutation screening in our group.Some articles have reported that compared with T.reesei,T.koningii,A.niger and P.decumbens T.orientalis EU7-22 shows higher activity of cellulase(exo-glucanase,endo-glucanase and P-glucosidase)and xylanase,and the corresponding genes also have been cloned and classified.In this study the transcriptional regulators ACE3 and BglR were cloned in Trichoderma orientalis EU7-22.The knockout strains Aace3 and?bglr were constructed to explore the fuction of these transcriptional regulators.(1)The function of positive transcriptional regulator ACE3 was investigated for cellulase and hemicellulase production in the strain.In this study the ace3 knockout strain Aace3 was constructed by homologous recombination in T.orientalis EU7-22.In the mutant strain,the filter paper activity,exoglycanases activity,endoglucanase activity,P-glucosidase and extracellular protein concentration were decreased to 20%,0.14%,8.3%,1.8%and 20%respectively of the parent strain.The transcription level of cellulase and hemicellulase genes also decreased observably.When cultivate on the avicel and CMC-Na plates the ?ace3 mutant grew slower and produced fewer spores than the parental strain.(2)The bglr gene of EU7-22 was cloned and sequenced by TAIL-PCR,the bglr disruption strain ?bglr was constructed.The protein and cellulose secretion level of this strain was investigated.It was shown that the BglR disruption in EU7-22 affect the cellulase activity.Compared with the EU7-22,the P-glucosidase activity is reduced by 47%.While the FPA,CBH and XYN activities were increased by 39%,22%,and 16%respectivly.From these results it is proposed that BglR is an up-regulate transcriptional factor of ?-glucosidase in EU7-22.(3)The cbhl gene of T.reesei was chosen as the promoter and the hph gene was selected as the selective maker.The expression cassette was successful constructed by fusion PCR.Which provide preliminary work to enhance the cellose ability of T.orientials EU7-22.