Cloning And Expression Analysis Of Three Manganese Peroxidase Genes From Lenzites Gibbosa

The white rot basidiomycetous fungi have a remarkable ability to decompose the most refractory lignin in wood. Decomposition of the lignin is possible due to the particular enzymes including lignin peroxidases, manganese peroxidases, versatile peroxidases and laccases. The white-rot fungus Lenzites gibbosa was isolated from rotted wood in Changbai Mountain. Recently, the cultural characteristics from L. gibbosa, and its decolorization ability have been studied. A novel MnP-encoding gene and two laccases were described, and the heterologous expression system in Pichia pastoris and Aspergillus nidulans were established successfully. This study presents the cloning and characterization of three manganese peroxidases genes of L. gibbosa. The research conclusions of the present study are as follows:1. Using RT-PCR, RACE to obtain three mnp gene fragments from L. gibbosa, and Genome walking-PCR was using to obtain the full-length genes of three mnp were obtained then named Lgmnp1, Lgmnp2, Lgmnp3(GenBank accession numbers:JQ327834, JN571114, JN571116), respectively. The three mnps are all interrupted by five introns. Accordingly, gene Lgmnp1consists of a1,095-bp open reading frame (ORF). The deduced amino acid sequence of the prepropeptide is364aa long. Lgmnp2has a1,098-bp-long ORF. The amino acid sequence of the prepropeptide encoded by Lgmnp2is365aa long. The ORF of Lgmnp3is1,101-bp-long. Lgmnp1cDNA shows the highest degree of identity with mnp from Trametes versicolor (AJ745879) which is up to83%homology, whereas Lgmnp2shares the highest similarity to mnp from T. versicolor (AJ745879) which is up to80%homology, and Lgmnp3have83%homology compare with mnp from T. versicolor (AY677129).2. Quantitative Real-time PCR experiment was using to compare the relative expression levels of Lgmnp1, Lgmnp2, and Lgmnp3. The transcript abundance for each isoform varied, depending mostly on the time after nutrient exchange. Lgmnp1is obviously influence by manganese concentration on any times, but Manganese availability did not influence the transcript level for Lgmnp2and Lgmnp3on any treatments. The transcript abundance of Lgmnp1is obviously higher than Lgmnp2and Lgmnp3after4days. Under high Mn2+treatment, Isoform Lgmnp1transcripts were expressed at levels approximately18.5-fold higher than Lgmnp2transcripts at8day by and48-fold higher than Lgmnp3transcripts at6day. Lgmnp2transcripts were expressed approximately3to5-fold higher than Lgmnp3transcripts under any experimental treatment.