Preliminary Study On The Function Of Bglr Gene In Myceliophthora Thermophila

Lignocellulose,as one of the main components of the plant cell wall,is the most abundant renewable biomass on the earth.Bioconversion of lignocellulose by cellulase to fermentable sugars is futher utilized by microorganisms to produce cellulosic biofuels,which has great significance for solving problems such as energy crisis,environmental pollution and achieving sustainable development.?-glucosidase plays a key role in the hydrolysis of cellulose and is a rate-limiting enzyme in the metabolic pathway of cellulase.It has been widely applied in various industrial fields such as food,pharmaceutical,bioethanol and rare oligosaccharide production.Myceliophthora thermophila is a thermophilic filamentous fungus that offers a large number of plant cell wall degrading enzymes that degrade cellulose efficiently.In the present research,we use homologous overexpression and RNA interference technologies to study the function of ?-glucosidase transcription regulator Bgl R and its effection of cellulase activity and expression of related cellulase genes in M.thermophila.(1)Overexpression of Mtbglr gene:The full length of bglr gene was cloned from M.thermophila ATCC42464 and the SLIC was adopted to construct Mtbglr overexpression vector.The promoter of MtPpdc and terminator of Mt Tpdc were used for construction of recombinant plasmid of overexpressing bglr gene.By transforming into M.thermophila,the positive transformant Mt8 strain was obtained successfully.The gene expression and ?-glucosidase activities were observed by realtime quantitative PCR and enzymatic determination.The results showed that the Mtbglr gene could increase the activity of ?-glucosidase under different carbon source under inducting conditions.Among them,1% corncob powder is the best inducing condition.The highest ?-glucosidase activities of Mt8 and WT were 2.27 U/m L and 1.35 U/m L,respectively,which were higher than those under other carbon sources.At the same time,the expression levels of the main cellulase genes bgl1,bgl2,bgl3,egl3 in Mt8 were 3.5,2.8,2.1,and 1.6 fold higher,respectively,than those of WT.The result indicated that overexpression of Mtbglr gene can increase ?-glucosidase activity and promote expression of the main cellulase genes.The above results showed that 1% corncob powder is an ideal inducing condition for cellulase production.(2)RNA interference of Mtbglr gene: In this study,the si RNA sequences of Mtbglr gene were designed.The promoter of MtPpdc and terminator of MtTpdc were adopted to construct Mtbglr RNAi vector.After protoplast transformation and PCR verification,the positive transformant Mt14 strain was obtained successfully.The gene expression and ?-glucosidase activities were observed by real-time quantitative PCR and enzymatic determination.The results showed the ?-glucosidase activities of WT and Mt14 were 1.31 U/m L and 0.71 U/m L on the 8th,respectively under 1% corncob powder inducing culture.At the same time,the expression levels of the main cellulase genes bgl2,bgl3,egl1,egl3 in Mt14 were 20%,48%,20% and 59% respectively,of those of WT.It indicated that interference of Mtbglr gene can reduce the ?-glucosidase activity and inhibit expression of the main cellulase genes.In summary,Mt Bgl R plays important roles as a positive regulatory in the regulation of cellulase genes expression in M.thermophila,which laid the theoretical foundation for the regulation of cellulase genes of thermophilic fungi.