Molecular Cloning And Sequence Analyzing Of Gene Encoding A Manganese Peroxidase From Schizophyllum Sp.F17

Mn-dependent peroxidase (manganese peroxidase, MnP), a kind of extracellular heme glycoprotein enzyme secreted by white-rot fungi, is a common lignin degradation enzyme. It can degrade many aromatic compounds when there is H2O2and Mn2+. In recent decades, due to the unique ability of the enyzyme to degrade many aromatic compounds, it has become a hot topics in food industry, paper industry and environment protection. The laboratory has already isolated and purified a strain of Schizophyllum sp.F17. In our previous studies, MnP from F17has shown potential for dye decolorization. So far, the studies are mainly focused on its cultural characteristic, decolorization ability and purification of the enyzyme, quite a few researches is reported on the gene cloning and sequence analyzing. In this paper, the extraction methods of total DNA and total RNA were investigated, at the same time, the the consensus fragment of MnP was cloned sequence analyzed. The main research results are listed as follows:The study of total DNA extraction methods from Schizophyllum sp.F17:in this experiment we uesd fresh mycelia and lyophilized mycelia as raw material and extracted total DNA by using glusulase exreaction and kit extraction, and then DNA were detected with agarose gel electrophoresis and ultraviolet absorption spectrum. The comparison and analysis of the purity, concentration and intergrity of the extracted DNA, it showed that glusulase-exreaction using lyophilized mycelia as raw material was the best method of extracting total DNA:better integrity, A260/A280ratio was between1.8and2.0, and high purity, the concentration was0.90ug/ul, and the whole experiment was simple, low cost and high efficiency.The study of total RNA extraction methods from Schizophyllum sp.F17:In order to seek a effective method of exteacting total RNA from white-rot fungi, Trizol Kit exreaction, CTAB-LiCl extraction and Diatomite-STE extraction were used and the extracted RNA were detected with agarose gel electrophoresis and ultraviolet absorption spectrum. The comparison and analysis of the operation process, concentration, purity and integrality of the extracted RNA showed Diatomite-STE extraction was the bestl method of extracting total RNA from white-rot fungi:28S rRNA,18S rRNA and5.8S rRNA were clear and intact, A260/A280ratio ranged between1.7～2.0, A260/A230was above2.0, and the yield of total RNA was up to1.102ug/ul; and the results also demonstrated that the RNA isolated by Trizol Kit showed obscure and absence, the yield of total RNA isolated by CTAB-LiCl was too low. And the step of precipitating RNA was improved, RNA was precipitated by NaAc-anhydrous alcohol after LiCl-anhydrous alcoho to remove the high purity of polysaccharide, and finally obtained high quality total RNA.Cloning and analyzing consensus fragment of MnP:degenerate PCR primers were designed according to the consensus sequence of some Mnp genes registered in GenBank by Clustal W matching and some other references, and finally primer P:(upstream:5’GGHGGTGCCGATGGSTC3’, downstream:5’TCRGACTGGAG SCGCATC3’) were successfully amplified a745bp fragment of MnP by degenerate PCR, and finally got a590bp fragment by intron shearing rules:AT:47.46%, GC:52.54%. As the result, the590bp gene sequences have been proved to be consensus fragment of MnP genes by comparing with other MnP genes by BLASTn, and exhibited the highest identity (88%) to that of Ceriporiopsis sp. MD-1(Accession-No.:AB362389.1). Further on, the predicted196amino acid sequences showed highly conservation and common characteristic of the plant-peroxidase-like family.