Functional Study Of The New Regulatory Factor HYF Of Lignocellulose Degradation In Trichoderma Reesei

As a large agricultural country,more than one billion tons of crop straw has been produced per year in China.With the continuous advancement of science and technology,most of the straw has been effectively utilized,but about 30%of the crop straw is still discarded or incinerated,causing serious environmental pollution and waste of resources,which limits the sustainable development of agriculture in China.Therefore,the rational and full use of crop straws can not only solve the environmental pollution caused by discarding and incineration,but also provide important raw materials for the exploitation of biomass in China,which in turn will benefit ecological sustainability.Extracellular lignocellulose degrading enzymes secreted by filamentous fungi are the results of co-regulation of various transcription factors.Although many of these regulatory factors have received widespread attention and were well studied,there are still undiscovered and unstudied regulatory factors.In the previous study of our lab,a new transcription factor was found in Aspergillus fumigatus Z5,transcriptomic results induced by different carbon sources indicate that this gene was regulated by the major components of xylem fibers(xylan and cellulose).In this study,we selected Trichoderma reesei QM6a as a research object which has strong ability to secrete cellulase and hemicellulose,then studied the new transcription factor,HYF.Analyzed the results and found that hyf gene was also regulated by cellulose which is the main component of lignocellulose.To further study the function of this gene,we constructed a knockout strain and an overexpression strain of this gene.Then,under the induction of cellulose,the only carbon source.The extracellular lignocellulose-degrading enzyme activities and the secretion of extracellular proteins were compared with wild-type strain.The main findings are as follows:1.Construction of knockout fragment,protoplast preparation and the improvement of transformation efficiency were preliminarily explored in Trichoderma reesei.Finally,the knockout strain of hyf gene was successfully obtained.2.Construction of overexpression fragment,selection of strong promoter and methods of transformant selection were initially investigated in Trichoderma reesei,and finally overexpression strain of hyf gene was successfully obtained.3.Knockout strains and overexpressing strains were used as materials to study the function of hyf gene.In this experiment,cellulose was used as the sole carbon source for liquid fermentation.Through the comparison of its enzyme activity and secretion of extracellular protein,it was found that this regulatory factor is related to the degradation of lignocellulose and is a downward regulatory factor in induction of cellulose;For knockout strains,activities of the extracellular endo-cellulase and xylanase in the knockout strain QM6a-?hyf were significantly higher than those of original strain at the early stage of cellulose induction.For the overexpressed strain QM6a-OEhyf,SDS-PAGE results showed that there was an obvious and relatively high content of the distinct protein at a molecular weight of 35 KDa-40 KDa,both in induction of carbon-free and cellulose as a carbon source,the original strain does not have the protein band;At the same time,in the process of cellulose induction,the activity of extracellular lignocellulose degrading enzymes of strain QM6a-OEhyf was significantly lower than that of the original strain.These results laid the foundation for further research on the function of the hyf gene.