Cloning And Function Analysis Of The Laccase Transcriptional Factor Gene Ltf4 From Pleurotus Ostreatus

As a large wood root fungi,Pleurotus ostreatus can secrete laccase to degrade lignin existed in culture material.Currently,12 putative laccase coding genes have been found on the P.ostreatus genome,among which poxc(lacc10)has been reported to encode a typical laccase.The expression of poxc gene always kept high level throughout the growth and development stages of P.ostreatus.Using the P.ostreatus poxc core promoter as bait,the laccase transcriptional factor coding gene ltf4 was obtained from the c DNA library of P.ostreatus fruiting bodies by using yeast one-hybrid technology.In this study,the ltf4 gene of P.ostreatus New 831 was characterized from the following aspects.Firstly,the CDS of ltf4 gene was cloned and analyzed by bioinformatics.Secondly,the expression patterns of ltf4 and poxc gene were analyzed by using RT-PCR.Thirdly,the ltf4 gene was heterologously expressed in Escherichia coli and the expressed LTF4 protein was purified by Ni-ion affinity chromatography.Fourthly,the in vitro interaction between LTF4 and the poxc core promoter was analyzed by electrophoretic mobility shift assay(EMSA).Finally,the ltf4 gene overexpression vector and antisense silencing vector were constructed here,which will be applied to identify the exact function of ltf4 in vivo.The main results of this study are concluded as follows:1?The CDS of ltf4 gene,which is 1,608 bp coding for a protein of 535 amino acids,was successfully cloned by using the fruiting body c DNA as template.The homologous alignment showed that LTF4 was highly homologous with a putative protein(KDQ29010.1)from P.ostreatus PC15,showing 97.53% identity.The result of protein structure prediction showed that LTF4,possessing a typical HTH-DNA binding domain,was belonged to the Moc R transcriptional factor family.2?The prokaryotic expression vector p EASY-E2-ltf4 was constructed to over express ltf4 gene in E.coli.Then expressed LTF4 protein was purified by using the method of Ni-ion affinity chromatography and analyzed by SDS-PAGE.The SDS-PAGE analysis showed that the molecular weight of LTF4 was about 60 k Da,which was consistent with the expected result.3?The expression patterns of poxc and ltf4 gene at the mycelium stage,primordial stage,morula stage,coral stage and fruiting body stage were determined through semi-quantitative RT-PCR(sq RT-PCR)by using the ?-actin coding gene as the reference gene.The results showed that both poxc and ltf4 gene were highly expressed at the mycelium knot stage.4?The interaction between LTF4 and poxc core promoter was analyzed by EMSA.The EMSA result showed that the LTF4 was able to bind to the poxc core promoter in vitro.5?Using the binary expression vector p Po-GPD constructed in our laboratory,the ltf4 eukaryotic overexpression vector p Po-GPD-ltf4+ and antisense silencing vector p Po-GPD-ltf4-were constructed by using the method of enzyme digestion and enzyme ligation,which were then transformed into Agrobacterium tumefaciens EHA105.And the transformation of the above vectors into P.ostreatus New 831 is being done by the method of A.tumefaciens-mediated transformation,which lays the foundation for the function analysis of ltf4 gene in vivo in the near future.