Preliminary Study Of Histone Modification On The Expression Of T. Reesei Cellulase

In order to investigate the epigenetic mechanism of histone modification on the expression of cellulase in Trichoderma reesei(T.reesei),the gene of histone H3 lysine methyltransferase(HKMT)and histone deacetylase(HDAC)were knocked out.And the mutant strains ?hkmt and ?hdac were successfully selected.The gene gcn5 of histone acetylase(HAT)was blocked by siRNA.And T.reesei-gcn5-T10 strain was successfully selected with better interference effect.The changes of mycelium of mutant ?hkmt and ?hdac and interfering recombinant strain T.reesei-gcn5-T10 were observed under microscope.And cellulase activities with filter paper and Carboxymethyl Cellulose-Na enzymatic activities of the stains were examined.Also,the mRNA expressions of cellulase gene cbh1,egl1 and activator gene xyr1 in the mutant strains and interfering recombinant strain were analyzed by Real-time fluorescent quantitative PCR(RT-qPCR).Also it was uesed to analyzed the relationship between hkmt,hdac,gcn5 and the expression of cellulase.The results showed that(1)the hyphae of the mutant ?hkmt and ?hdac were observed to be longer than the starting strain T.reesei QM9414 under the objective lens of the same magnification,and the branches also were longer than those of the control.While the hyphae that interfere with the recombinant strain T.reesei-gcn5-T10 was shorter and thicker than the starting strain T.reesei QM9414,and the ends were enlarged and the inside became turbid.(2)The FPA and CMCA of the mutant ?hkmt and ?hdac were significantly higher than those of the starting strain T.reesei QM9414.The mutant ?hkmt were 5.00 IU/mL and 15.00 IU/m L higher than the starting strain T.reesei QM9414,respectively.And the mutant ?hdac were 6.50 IU/mL and 15.00 IU/mL higher than those of the control,respectively.While the FPA and CMCA of the recombinant strain T.reesei-gcn5-T10 were significantly lower than those of the control,which were 10.0 IU/m L and 5.2 IU/mL,respectively.(3)RT-qPCR showed that the relative expression levels of cellulase gene cbh1,egl1 and enzyme activator xyr1 in the mutant strain ?hkmt were higher than those of T.reesei QM9414,which were 4.51 times,3.87 times and 2.51 times respectively.The expression levels of cbh1,egl1 and xyr1 in ?hdac were higher than those of the control,which were 6.50 times,6.01 times and 4.51 times respectively.While in the recombinant strains T.reesei-gcn5-T10,the expression level of gene cbh1,egl1 and xyr1 were lower than those of the control,which were 0.34 times,0.84 times and 0.44 times of the original strain respectively.(4)RT-qPCR analyzed the relationship of hkmt,hdac and gcn5 showed that interference of gcn5,that is,the decrease of HAT expression,resulted in the increase of hdac expression and the increase of hkmt expression.Conclusion: The methylation and deacetylation of the histone were inhibited when the genes encoding hkmt and hdac were knocked out respectively,the expression level of the cellulase gene cbh1,egl1 and the activator factor xyr1 were significantly increased,and eventually activating cellulase expression.While silencing gcn5 inhibited histone acetylation and the expression of the genes were significantly decreased,leading to decreased cellulase activity.