| White rot fungi,the important participants of elements cycle in nature,can completely decompose lignocellulose into H2O and CO2.Although white rot fungi are abundant and widely distributed,there are still big doubts about the mechanism of wood degradation.In this thesis,a white rot fungus,Phlebia tremellosa BBEL0901,which can selectively degrade lignin,is used as the research material.Based on sequencing and analysis of its whole genome,the genes involved in lignin degradation were further excavated and analyzed by bioinformatics methods,with a view to clarifying the genetic information of the lignin degradation of this strain.The acquirement of monokaryons is of great importance to whole-genome sequencing of white rot fungi,therefore,to obtain sufficient protoplasms,we firstly optimized the conditions for protoplasm preparation of strain BBEL0901 by lysozyme method based on single-factor experiments combined with orthogonal experiments.Monokaryons were screened from the regenerated protoplasms via nuclear fluorescence staining,ITS sequence analysis and heterogeneity assay.Whole-genome sequencing of the selected monokaryon was performed and then,the sequences obtained by second-generation and third-generation sequencing were evaluated and subsequently analyzed.The main research contents and results of the paper are as follows:1.The effects of mycelial age,enzymolysis time,lysozyme concentration,rotation speed,type and concentration of osmoticums were investigated through single-factor experiment,on the basis of which,the condition of protoplasts preparation from strain BBEL0901 was optimized based on orthogonal experiments.The optimal condition was:mycelial age was 4.5 days cultured in enriched PDB medium,NaCl solution(0.65 mol/L)was used as osmotic pressure stabilizer,and the protoplasts were collected by centrifugation at 3000 r/min after enzymatic enzymolysis with 5%lysozyme for 3 hours.Nuclear staining with DAPI was used to screen the regenerated colonies from protoplasts.Three protoplasted monokaryons,M73,M74 and M81,were obtained and strain M73 was further confirmed to be monokaryon by ITS sequencing and Kmer curves.So strain M73 was finally selected for whole-genome sequencing.2.Both the second-generation and third-generation sequencing technologies were used to sequence and assemble the whole genome of the monokaryon M73 43 scaffolds were assembled from the raw sequence data of 8.23G and the whole genome size was 40.7 Mb,with a sequencing depth of 133 ×.The GC content was about 51.41%,and a total of 12,362 coding genes,272 tRNAs and 18 rRNAs was annotated.Phlebia tremellosa had an approximately complete degradation system of cellulose,hemicellulose and pectin with low copy number of key enzyme genes.A complete lignin-degrading system included 86 genes related to lignin degradation,13 annotated pathways involved in lignin degradation,and more than 4 metabolic pathways for lignin and its intermediates.326 cytochrome P450 genes were identified,which was more than that of most reported white rot fungi.3.The conserved domains,evolutionary relationship,promoter cis-elements and the primary,secondary and tertiary structures of laccases,manganese peroxidases and lignin peroxidases were further analyzed in this part.The molecular weight of laccases,lignin peroxidases,and manganese peroxidases in Phlebia tremellosa was between 55.69～106.67 kDa,38.21～45.36 kDa,and 40.08～40.51 kDa.Most of the ligninases were located outside the cell,and some were located on the cell membrane.Five of the six laccases had copper-binding sites,and all the peroxidases had heme binding sites.The genetic distance of the genes of lignocellulolytic enzymes were closer to the white rot fungi Phlebia chrysocreas and Phlebia brevispora.There were multiple kinds of cis-acting elements upstream of its binding location.In summary,Phlebia tremellosa has a complete enzyme system involved in lignocellulose degradation,a strong lignin-degrading system and a rich lignin metabolic pathway.The results provided an important reference for further understanding the molecular mechanism of selective lignin degradation by Phlebia tremellosa. |
