| Coriolopsis gallica is a kinds of white rot fungus belongs to genus Coriolopsis family Polyporaceae.As a widespread white-rot basidiomycota,they are able to degrade lignin and a variety of phenols and aromatic amine compounds,which leads to broad application prospects in biomass utilization,environmental protection,paper industry,food industry and so on.In previous study,an high-production laccase C.gallica like strain,named T906,was obtained using mutagenesis.Mutant T906 show inheritable variations in morphological traits,growth traits and metabolic patterns,but the internal mechanism still unkonwn.In this study,the physiological and biochemical characters between wild strain and mutant strain were analyzed.The differentially expressed unigene and metabolic patterns between two strains were also analyzed in detail based on RNA-seq.Five laccase isozymes from two strains were also cloned and analyzed.Analysis of ITS from mutant T906 and its parent TCK revealed 99.89%nucleotide similarity with C.gallica strain RLG9577RITS,which were belong to genus of Coriolopsis in the family of Polyporaceae.Hence,the mutant fungi and its parent has been named as C.gallica T906 and C.gallica TCK,respectivly.The colonie of mutant T906 was smaller but thicker than that of the parent strain TCK.The mycelium of mutant strain T906 was stronger and more regular than that of parent strain TCK when cultured on PDA solid medium.The cell wall of the mutant strain was significantly thinner than that of the parent strain by transmission electron microscope.And numerous vacuoles were accumulated in cytoplasm of mutant T906 along with the prolongation of the culture time.The biomass of the mutant strain was significantly increased than that of the wild strain in the optimized lipid fermentation medium TNI during the incubation period（1.09-1.65 times）.To investigate the transcriptomic variation between wild and mutant fungi,mycelia samples collected at 3 day and 8 day were used for RNA-Seq analysis,respectively.A total of over 53625112 clean reads and 4,826,260,080 nt data were obtained with Q20 above94.5%.The sequence length distribution patterns were similar and no bias among four samples.After filtering for adaptor sequences,duplication sequences,and low-quality reads,a total of 25199 unigenes with average length of 908 nt were assembled,and 20788 of them（82.50%）contained open reading frames（ORFs）.Of the 25199 unigenes,20018（79.44%）had significant hits in public nucleotide or protein databases.59.6%of the hits annotation were from dichomitus squalens and 31.30%were from Coriolus versicolor.While 770unigene hadn’t matching sequences in all the public sequence databases,which maybe represented the new genes or the errors assembly fragments.Functional clustering results showed that there were 7664,8434,and 12426 unigenes can be annotated into 44 GO terms,26 COG cluster,and 108 metabolic pathways,respectively.Based on the normalized FPKM（Fragments Per Kilobase of transcripts per Million mapped fragments）values,all the differentially expressed genes（DEGs）with FDR（False Discovery Rate）≤0.001 and log 2 fold-change≥1 between each pair of samples were identified,which resulted in a total of 8920 DEGs.Compared with the same period sample of wild strain,3024,3920 down-regulated DEGs and 967,1728 up-regulated DEGs were identified in 3 day sample and 8 day sample of mutant T906,respectively.There were 2426down-regulated DEGs and 1912 up-regulated DEGs between 3 day and 8 day samples of mutant,while only 654 down-regulated DEGs and 723 up-regulated DEGs between the samples of TCK at same period,which suggested the gene express pattern of mutant are more susceptible to fermentation time.Ribosome and protein translation involved terms were the most enriched GO term,followed by non membrane organelles（proteasome）and the synthesis and metabolism of basic material.The oxidoreductase activity GO term（especially oxidoreductase activity,acting on the CH-OH group of donors,NAD or NADP as acceptor）of the mutant strain also changed significantly accompanied by the extension of fermentation time.The pathway enrichment analysis supported the results of GO enrichment analysis,thus,the ribosome involved pathway were the most significant enriched pathway(P<2.15e-25),followed by Aminoacyl-tRNA biosynthesis,EMP and TCA pathway.The gene express level in these pathway were down regulated in mutant strain significantly.Followed with the extension of fermentation time,gene express patterns among Amino sugar and nucleotide sugar metabolism,starch and sucrose metabolism,proteasome metabolism,glucuronate pathway,and the secondary metabolism changed greatly in mutant,of which,the expression level of key genes have increased significantly.The mutant T906 had reduced the synthesis level of proteins,but improved the utilization efficiency of intracellular amino acids and proteins,reduced the utilization of intracellular glucose,but improved the utilization efficiency of extracellular sugar,as well as reduced the biosynthesis of fatty acid,but improved the Fatty acid metabolism.The mutant mitochondria oxidative phosphorylation becoming active,which lead to excessive ROS and oxidative stress increased.The signal transduction pathway,include MAPK and PKA,were also changed significantly in mutant T906,resulted in the external signal can be quickly transmitted and respond.The mycelium protoplast of mutant strain and wild strain at 3,5,8,11 day were prepared and used for the study of cell cycle,cell apoptosis,cell autophagy and ROS level using flow cytometry（FCM）technology.The results revealed that the cell number of G2/M phase of mutant strain T906 increased obviously,which suggested the mutant strain had accelerated the progress of cell division.At the same time,both of the number of apoptotic cell and autophagic cell increased in mutant strain accompanied by the extension of fermentation time.Reactive oxygen species（ROS）analysis showed that the ROS level in mutant cells were significantly increased compared with that of wild cells,especially in the late stage of fermentation.And the mutant cells increased the expression level of ROS scavenging enzymes,resulting in enhanced tolerance to oxidative stress.There were 5 laccases,named laccas1-5（Lcc1-5）,were cloned and analyzed from C.gallica.The gene of 5 laccases fom C.gallica ranged from 1667 nt to 2388 nt,encoding527-528 amino acid transmembrane protein with predicted molecular weight of55.50-56.78 kDa.Phylogenetic analysis showed that all of the five laccases have remarkable homolog in published databases.All of the five laccases protein included 3copper ion binding domains and 3 domain coupling regions,which were corresponding with the conserved pattern of laccase.The results of quantitative analysis showed that the Lcc1 transcript level in the T906 were accounted for 93%（3 day）and 98.53%（8 day）of the total laccase transcript,and this proportion were 41.48%and 74.37%in the wild strain,respectively.In mutant cell,the expression level of Lcc1 gene was much higher than that of the wild cell during the whole fermentation progress（14.25 to 15.50 times）.There were 11nucleotides and 4 amino acid mutation between LCC1 from mutant strain and wild strain,which do not effect the protein structure and function. |
