Studies On Transcriptional Regulation Factors Of Lignin Peroxidase Gene (lipC) Of Phanerochaete Chrysosporium

Tie white-rot basidiomycete-Phanerochaete chrysosporium is an excellentmodel organism for studying the mechanism of lignin biodegradation. Due to theexcellent ability to degrade lignin, as well as related compounds found in explosivecontaminated materials, P.chrysosporium is greatly potential in bio-pulping andenvironmental protection. The extracellular ligninolytic system is a complicatedprocess and comprises many enzymes, including lignin peroxidases and manganeseperoxidases. Northern blot and RT-PCR analyses revealed that the transcription oflips and mnps are regulated by nutrients such as carbon and nitrogen, etc. Theprevious investigations showed that two nuclear protein binding sites, named PBE1and PBE2, located upstream of the transcription initiation site of the lipC gene.Therefore, it is very important to clone the genes encoding the transcriptionalregulators of lip genes expression.First, the yeast one-hybrid was performed by screening 3-day cDNA library ofP. chrysosporium, using PBE1, PBE2 and PBE1+PBE2 as bait, respectively. Theresults suggested that there were 9, 6 and 10 candidate clones related to regulatinglipC transcription by targeting PBE1, PBE2 and PBE2+PBE2, respectively.Bioinformaties analysis revealed that only 4 clones are localized in nucleus, namedPBE1-A12.4, PBE1-A20.3, PBE2-A5.1 and PBE1-2-A27.1. Sequence analysesrevealed that PBE1-A20.3 is an unknown protein. PBE1-A12.4 has high homologywith 14-3-3 protein. The 14-3-3s are important in the regulation of such crucialcellular processes as apoptosis, cell division, signal transduction, protein trafficking,transcription. The 14-3-3s usually play roles via direct protein-protein interactions.Previously, 14-3-3 proteins were characterized a cruciform DNA-binding activity inhuman, plant and yeast. The interactions are involved in DNA replication. Functionsites analysis suggested that the clone PBE2-A5.1 contains an ATP binding site and a leucine zipper pattern. The pattern is present in many gene regulatory proteins, suchas: the CCATT-box and enhancer binding protein (C/EBP), cAMP response element(CRE) binding proteins, etc. PBE1-2-A27-1 encoded a protein containing, histidinetriad (HIT) domain. Proteins in the HIT superfamily are conserved asnucleotide-binding proteins.To identify the interactions between the positive clones and cis-element, theORF of 14-3-3 was then subcloned and expressed in Escherichia.coli using pET28aexpression vector. SDS-PAGE analysis showed that the recombinant protein exist asinclusion body and soluble fraction, with molecular weight 33 kD. Then the solubleproduct was purified with Ni-colunm. The purified protein could bind toPBE1+PBE2, labeled with ³²p by gel mobility shift assay (GMSA). The resultsshowed that the shifted band was enhanced when increase 14-3-3 protein and theshifted bands disappeared when 40×cold probe was added. These results indicatedthat 14-3-3 protein was able to specifically bind to the PBE1+2 probe.Secondly, the 14-3-3 gene was used to screen the 3-day library by yeasttwo-hybrid and more than 600 clones were obtained on the SD/-Ade/-His/-Leu/-Trpplates, but a few of positive recombinants could activate three reporter genes.Plasmids isolated from yeast transformants were successfully transformed into E.coli. These clones could be classified into 6 types on the basis of the digestionpatterns with Haeâ…¢and Sau3AI as well as their sequences. A PROSITE searchrevealed multiple potential phosphorylation sites in the 6 proteins, which arepotential binding sites of 14-3-3 protein. Two proteins containing predicted WDdomain could interact with 14-3-3. The amino acid sequence of Y2H147 is 27%similar to transcription initiation factor TFIID. The three-dimensional model of14-3-3 suggested that it contains 9 helix structures and may form homodimer in P.chrysosporium. Protein-protein prediction indicated that 14-3-3 protein could bindwith Y2H147, Y2H389 and Y2H412. These results suggested that 14-3-3 in P.chrysosporium may function in different cellular processes, especially in genetranscriptional regulation.