Expression Of MilRNA In Trichoderma Reesei And Its Regulation Of Cellulase Expression

Trichoderma reesei is an important cellulase producing strain.It's also a model strain for studying the regulation mechanism of cellulase synthesis in filamentous fungi.Moreover,the regulation mechanism of cellulase production in T.reesei has been extensively studied.Micro RNA(miRN A)is ubiquitous small RNAs that present in various organisms.They combine with target mRNA specifically to regulate gene expression.In recent years,micro RNA-like RNA(milRNA)has been identified by high-throughput sequencing in several filamentous fungi,such as Neurospora crassa,Sclerotinia sclerotiorum,Metarhizium anisopliae,Trichoderma reesei,etc.However,there are only few reports focusing on the functions of miRNA or milRNA in Trichoderma reesei till now.In this work,we studied the functions of 4 milRN As in T.reesei QM9414,based on the analysis of the expression levels of total 13 milRN As,which have been obtained by high-throughput sequencing of T.reesei QM9414 in our previous study,under inductive and repression media for cellulase production.We constructed the overexpression vectors that contained the overexpression cassette for milRNAs using the T.reesei pdc promoter and inhibition vectors that contained the Tough Decoy(TuD)sequence of the milRNAs,and transformed T.reesei QM9414 with these vectors.The transformants were selected and cultivated in cellulase production media.The relationship between milRN A expression level and cellulase expression level was analyzed through RT-qPCR and cellulase activity analysis,and the fuctions of the four milRNAs on regulation of cellulase production were verified.Finally we optimized the culture media of the recombinant strains which showed elvated cellulase productivity compared with the origin T.reesei strain to further improve the cel ulase activity.The result showed that milR4 and milR9 participated in the regulation of cellulase expression.The data of qPCR analysis showed that the TuD sequence decreased the expression level of milRNA,indicating that the TuD sequence has played an inhibiting effect,while the overexpression cassette enhanced the expression of the milRN A,indicating that the precursor sequence of milRNA was amplified successfully.The cellulase productivity of P-milR4 recombinant strain was decreased to 0.919 times of the original strain,while the cellulose productivity of TuD4 recombinant strain was increased to 1.228 times of the original strain.This result indicated that milR4 negatively regulated cellulase production.In addition,the activity of recombinant strain TuD9 was decreased to 0.946 times of the original strain,while P-milR9 was increased to 1.349 times of the original strain,indicating that milR9 positively regulated cellulase production.After optimizing the cellulase production medium,a recombinant strain P-milR9 with relatively high enzyme production was obtained,and its activity was 1.454 times of that of the original strain.In this work,we found 2 milRN As: milR4 and milR9 that could significantly alter cellulase activity of T.reesei by constructing the overexpression vectors and inhibition vectors.Furthermore,the cellulase activity of recombinant strain P-milR9 was further improved by optimizing the culture medium.This work provides a new understanding for studying the mechanism of miRNA regulation in T.reesei and demonstrates a new strategy and approach for increasing cel ulase activities in T.reesei.