Preliminary Study On The Interaction Mechanism Of Trametes Versicolor-F21a And Cyanobacterial Cells In Aquatic Environment

The outbreak of algal blooms not only lead to the death of fish and other aquatic organisms due to asphyxiation,but also endanger the health of human and other organisms via secreted-toxins directly.Compared with the physical and chemical methods,microbes based method for algae control is simple,less pollution etc.,and becomes a hot spot in control of cyanobacteria bloom.Currently a large number of researches concentrated on screening algicidal bacterial strains and cyanophage virus with excellent algicidal characteristics,while these methods have some harmful influence on the transparency and aquatic ecosystem.In recent years,a new method which utilize a new aspect of fungi to rapidly remove cyanobacteria bloom was discovered.A strain F21a of fungus T.versicolor with higher algicadal capacity was isolated.This fungus can degrade all of the algal cells in the fungus-alga co-cultrue system,resulting in clear and transparent of the waterbodies.However,the mechnism of this new method is still unclear.In this paper,T.versicolor-F21a was used as the tested strain to investigate the mechanism this new fungus-alga interaction via scanning electron microscopy and transcriptome sequence.Differently expressed genes of the fungus during co-culture with algal cells were enriched,and some of the important genes for decomposition enzymes were further verified by RT-PCR and enzymatic assays.Transgenic system of the fungus was established to verify the important genes in vivo.The main results are as follows:(1)Scanning electron microscopy was used to observe the co-cultured between the mycelia of T.versicolor-F21a and algal cells during different time.The results show that many less compact cavities were formed by the fungal hyphae to wrap the algal cells.The algal cells were deformed along the time,and completely disappeared in the end.(2)The fungus-alga co-culture samples during 0 hour,1 hour,and 6 hour were used for transcriptome analysis.The three samples obtained 5986250,6290837,and 5833802 clean reads,respectively.About 53%of the clean reads can be mapped to T.versicolor reference genome.Annotation and analysis of differentially expressed genes showed that 1439 genes of the 1 hour co-culture fungus are significantly up-regulated and 1981 down-regulated compared to that of the 0 hour,while 1161 genes in 6 hour fungal sample are up-regulated and 1887 down-regulated compared to that of the 0 hour.The differentially expressed genes(DEG)of co-culture was further investigated by GO term enrichment analysis.The results show that in biology process level,the DEG was mainly associated with cellular process,metabolic process etc.At the cell component level,the DEG was mainly related to cell part,organelle part,and macromolecular complex.At the molecular function level,the DEG was primarily related with binding,catalytic activity.KEGG Pathway Enrichment analysis shows that the DEG is mainly related to Cellular Processes,Environmental Information Processing,Genetic Information Processing,and Metabolism.In Cellular Processes level,the DEG is involved in Transport and catabolism,cell growth and death.In environmental information processing level,the DEG is mainly involved in signal transduction.In genetic information processing level,the DEG is involved in transcription,translation,Folding,sorting and degradation.Especially in the metabolic level,the DEG is mainly involved in Glycan biosynthesis and metabolism,lipid metabolism,biological degradation of harmful substances,energy metabolism,carbohydrate metabolism,amino acid metabolism.GO and KEGG enrichment analysis implies that the fungus T.versicolor-F21a probably decompose the components of cyanobacterial cells,and then transport the metabolites into its own cells as raw materials for growth and development.Decomposition and metabolic enzymes of T.versicolor fungus should play important roles during the algicadal process.The up-regulate genes were further annotated in CAZymes web,and result showed that majority of the degradation enzymes were cellulase,ligninase,manganese peroxidases,laccases,?/?hydrolytic enzymes etc.About54%percent of enzymes has secretory peptide,indicating that more than half up-regulated decomposition enzymes can be secreted into the cavity structure formed by fungal mycelia.Expression level of these hydrolase genes during the entire degradation process by semi-quantitative PCR showed that most of the accumulation of the single tested genes was positively related with degradation rate of algal cells.Principal component analysis further shows that hydrolase genes are not contributed equally during the degradation processes.Some genes,such as jgi|Trave1|126211(Cellobiohydrolase I),jgi|Trave1|30530(GH13),jgi|Trave1|60067(GH16),jgi|Trave1|132063(GH18)?jgi|Trave1|58303(?-glucosidase),jgi|Trave1|138531(laccase),jgi|Trave1|135205(GH16)?jgi|Trave1|68611(GH72),jgi|Trave1|131032|(GH5)might play a leading role.These genes should be the major degradation genes,which was consistent with the results of preliminary enzymatic essay.(3)The optimum conditions of protoplast preparation and regeneration for T.versicolor-F21a is cultivated for 4 days at 28?on the PDA medium,0.6 mol/L KCl is the optimal isotonic conditions,enzymolysis for 4 hours at 30?with mixed enzymes(2%cellulase+2%snailase+2%lywallzyme).In the optimal condition,the production of protoplast reach 2.375×10~7/mL,and the regeneration rate is 0.74%.The use of restriction endonuclease-mediated technology can transform pAN7-1 plasmids into the protoplasts of T.versicolor-F21a which were verified by PCR.Transfer of the mycelia for several generations showed that exogenous gene can be stably expressed in the transgenic strain.In this paper,multiple research techniques were used to study mechanism of T.versicolor-F21a efficiently eliminate algal cells,especially the molecular mechanisms.A large number of differentially expressed fungal genes during the algicidal process were observed.Enrichment analysis of the different expressed genes and later verification identified several key fungal enzymes for the breakdown of cyanobacterial cells,and preliminarily elucidate the biochemical basis of the fungus to wrap and eliminate cyanobacterial cells.The results of this paper will not only enrich our knowledge on fungi-cyanobacteria interaction,but also provide some clues in screening more efficient algicidal fungus,or even generate more efficient transgenic algicidal fungus.