Cloning And Heterologous Expression In Aspergillus Nidulans Of Genes Encoding Manganese Peroxidase From Hericium Erinaceum

The study on Hericium erinaceum (Bull.:Fr.) Pers. CB1strains were classified identification and phylogenetic analyses, its major ligninolytic enzymes were detected. Four different types dyes of Alizarin red, Neutral red, Congo red, Crystal violet were decolored treatment by enzyme liquid. Strain CB1for gene donor, cloning manganese peroxidase(MnP) isozyme genes Hemnpl and Hemnp2full length of cDNA genes and DNA genes, and through the fluorescence quantitative PCR Hemnpl and Hemnp2were detected differential expression,and then to construction recombinant plasmid,through the protoplast transformation methods two MnP isozyme gene transformation into the constitutive A. nidulans, They were obtained effectivee expression of two MnP isozyme Aspergillus nidulans engineering strain.The research conclusions of the present study as follows:1. Firstly, Cultural characteristic of H. erinaceum strain CB1was described. The results indicated that the appearance and microscopic characteristics of strain CB1and the color and forms of H. erinaceum fruiting body are very similar. The phylogenetic analysis of Hericium five species17different regional strains using the internal transcribed spacer (ITS) region, results showed that strain CB1and other strains from H. erinaceum were nearer in genetic distance and clustered together with them. The strain belongs to Hericiaceae, Hericium, Hericium erinaceum.2. The classification status of H. erinaceum strain CB1was fixed, Then,add the with different Mn2+concentrations and sawdust as the substrate by LNAS (Low Nitrogen Asparagine Succinic acid) culture solution in such situation,and its the major ligninolytic enzymes were detected.The results indicated that H. erinaceum could produce MnP and laccase simultaneously, but no LiP. Detection on different manganese ion as the substrate,The highest MnP activity were259.55U-L"1when sawdust was added to LNAS culture solution with600umol-L"1Mn2+substrates. Results indicated that Mn2+to MnP enzyme activity play induction.Mn2+was proved to be the essential factor for H. erinaceum to produce MnP,the lignin-degrading enzymes of the strain CB1and the relationships between enzyme activities and medium composition and substrates were gained. When the extracellular enzymes achieve optimum, the4different structure dyes add to the culture system for common culture, from H. erinaceum solid and liquid culture medium for dyes degradation decoloring, and results show that4dyes concentration5mg/L,20mg/L,50mg/L and100mg/L, Both alizarin red and neutral red are best decolorization effect, Congo red took second place, Crystal violet decolorization effect show the difficulty of degradation.3. Using degenerate PCR, RT-PCR, RACE, and Genome walking-PCR methods,etc. Molecular cloning of gene encoding two MnP isozyme genes from H. erinaceum. the full length cDNA were obtained then named He-mnpl and He-mnp2, respectively, He-mnpl and He-mnp2cDNA genes contains1080bp and1086bp,encoding for precursor polypeptide of359and361amino acids.Both two isozyme DNA and cDNA gene homology67%and81%,respectively. They genomes contain the11exon and10introns.Forecast and analysis amino acids of He-mnpl and He-mnp2by BioEdit、ClustalW2.1and3D software.4. The inequality expression levels of H. erinaceum two MnP isozyme genes were detected by quantitative Real-time PCR method. The results show that He-mnp1genes is the highest expression level for200μm/1Mn2+concentrations in10d, He-mnp2genes transcription level to reach the highest for600μm/1Mn2+concentrations in7d,He-mnp2gene expression than the highest amount He-mnpl gene expression highest amount was more than8times, Thus it can be seen that He-mnp2gene is strongly influenced by Mn2+.H. erinaceum MnP isozyme genes exist between different transcription regulation mechanism, He-mnp1and He-mnp2isozyme gene expression after reaching the highest level with time changes, regardless of the manganese ion concentration and increased induction time expression is restrained,He-mnp1and He-mnp2by Mn ion concentration of different process induction control.5. The coding complete MnP isozyme CDS gene, He-mnp1and He-mnp2were cloned by RT-PCR from H. erinaceum, and heterologous expression in A nidulans. the recombinant plasmid pLB01/He-mnp1and He-mnp2were constructed. And, using PEG/CaCl2protoplast transformation method, He-mnp1and He-mnp2were successfully transferred to auxotrophic stain TN02A7of A.nidulans. The two new tranformant stains, Auxotrophic stain TN02A7, WJA01and stain CB1were detected the MnP activity in the same shaking medium containing lignin,result showed that TN02A7-He-mnpl could produce MnP activity in the absence and presence of heme, but the MnP activity was up to38.31U-L-1on96h with heme which was8.64times higher than that without heme;TN02A7-He-mnp2could produce MnP activity in the absence and presence of heme, but the MnP activity was up to64.06U-L-1on96h with heme which was15.88times higher than that without heme,but tranformant stains produce MnP activity less than that of H. erinaceum CB1on144h, Because of TN02A7and WJA01could not produce MnP activity at any time, indicating that the gene He-mnpl and He-mnp2had been successfully transformed into A.nidulans expressed in lignin environment. The results of heme was one of restrictive factor for rescombinant mnp gene to express in A.nidulans.