Study On Proteomics Of Nematode Trapping Fungi-duddingtonia Flagrans

Duddingtonia flagrans,as a representative species of nematode-trapping fungus,has strong environmental tolerance and nematode-trapping ability,and is thought of as the most promising candidate of biological agents.At present,domestic and foreign scholars have done a lot of morphological observations,biological characteristics and clinical application experiments with the fungus.However,there is still short of information about the nematode-trapping mechanism,and the study of the molecular background and nematode-trapping mechanism of the fungus can provide a theoretical basis for the development of efficient and stable biocontrol agents.In view of this,D.flagrans,a nematode-trapping fungi with great biological control application prospects,was selected and extraction method for mycelia proteins of the fungus was optimized;Then proteomes of different predation periods were analyzed to determine the changes of the mycelium at the levels of protein by using iTRAQ technology,and some proteins related to the nematode trapping were discovered and verified by quantitative PCR.From the protein level,the trapping process of D.flagrans was revealed,which was very useful to enrich our understanding of nematode-trapping fungi and reveal the molecular mechanisms of the nematode-trapping process.The main research results were as follows:(1)The urea-thiourea lysis method,snail enzyme digestion method,protein extraction kit and TCA-acetone-SDT lysate method were used to extract D.flagrans mycelial proteins respectively.Based on the results of SDS-PAGE and Bradford concentration test,it was found that the protein extraction kit combined with liquid nitrogen grinding was the best method for preparing intracellular proteins of D.flagrans,but for proteomics research,TCA-acetone-SDT lysate method was better.(2)The process of trapping nematode by D.flagrans was observed by electron scanning microscopy.The trapping process was divided into three stages: the earlier stage of capture(0h),the middle of capture(12h)and the later stage of capture(48h,72h),and the fungal samples were collected at these time points respectively.The D.flagrans mycelia induced by distilled water was used as the control group(DF group),and the D.flagrans mycelium induced by the Caenorhabditis elegans extract was added as the experimental group(CE group),and iTRAQ technology was carried out for qualitative and quantitative analysis.The results showed that 4244 proteins were identified.Among the control group,compared with group of 12 h,the number of differential proteins in group of 72 h was the highest,with a total of 572;For group of 48 h and 0h,the differential proteins identified were the least,only had 144 proteins.In the experimental group,there were 474 differential proteins identified between group of 12 h and 0h,and there were 27 differential proteins detected between goup of 72 h and 48 h.At the same time point,compared with the control group,there were 296 differential proteins identified in group of 48 h,and 104 proteins were identified in 72 h.The GO function of the differential proteins in each treatment group was analyzed.The results showed that the differential proteins in the fungus related catalytic activity,molecular binding function,transport activity,metabolic process,biological regulation,stress response,biolocation,carbon utilization,and substance synthesis showed significant different.Through KEGG enrichment analysis,it was found that the differential protein mainly involved in ubiquitin-mediated proteolysis,endoplasmic reticulum protein processing,sphingolipid metabolism,adhesion process,MAPK signaling pathway,AMPK signaling pathway,energy metabolism and carbon metabolism,peroxidase,oxidative phosphorylation and other eight categories.These pathways were related to the process of the fungus-feeding nematode.Through the above analysis,it was predicted that such as neutral ceramidase,sphingomyelin phosphodiesterase,tyrosinase,might play key role in the predation process.These findings would supply the foundations for revealing the biological processes of the fungus,fungal action on nematodes,and screening and identification of predation-related proteins,which was very helpful to elucidate of the nematode-trapping mechanism of D.flagrans in future.(3)According to the results of iTRAQ analysis,12 kinds of target proteins related to nematode-trapping were selected,and the results of fluorescence quantitative PCR showed that more than half of the genes expression results were consistent with iTRAQ results,which indirectly demonstrated the accuracy of proteome sequencing and analysis was reliable.