| Duddingtonia flagrans is one of the most well-studied and widely utilised nematode-trapping fungus,as it has high environmental tolerance and can pass through gastro-intestinal tract of grazing livestock,without reducing its germination rate and the efficiency of predation Therefore,D.flagrans has been widely used as a biological agent in prevention and control of animal parasitic nematodes.So far,D.flagrans has been studied on morphology,biology and evaluation of clinical use.But little is known how it possessed the ability to kill nematodes and its trophic modes at the molecular level.In order to investigate the features and advantages of D.flagrans in control of nematodes,the draft genome and transcriptome of D.flagrans were sequenced and studied by comparative analysis.These research provided a series of differently expressed genes related to nematode-trapping and some of the genes were validated by real-time quantitative PCR.Besides,the interaction between D.flagrans and infective larvae of nematodes was checked with optical microscope as well as scanning electron microscope.In order to provide the research material for the study of gene function using gene knockout technology,the protoplast-producing efficiency of D.flagrans was evaluated by fluorescence labeling.The main research achievements were as follows:(1)The method of genome DNA extraction of D.flagrans was explored,and the result revealed that the DNA extraction method of solid culture systems with dialysis membrane combined with spin columns was simple and feasible.Then genome survey was carried out to determine the research strategy which was fit for the fungus.The result indicated the average genome size of D.flagrans was about 37.6 Mb and GC content was as high as 44.95%with low hybridity percentage.The genome survey has laid a theory foundation for the detailed genome mapping of nematode-trapping fungus-Duddingtonia flagrans.(2)After deep sequencing by SMRT technology,we have used comparative genome analysis to describe the genome features and potential pathogenicity of D.flagrans.The compact genome size and absence of RIP mechanism revealed the slow evolution of D.flagrans.Likewise,homology analysis and gene family analysis also suggested its high conservation of genome.For pathogenicity,D.flagrans had abundant potential genes involed in nematode trapping and virulence as well as infection,such as PHI genes,Cytochrome P450 genes and various protease coding genes,suggesting its high pathogenicity.In contrast,relatively fewer carbohydrate-degrading related genes showed weaker saprophytic capability of D.flagrans.(3)The observation of interaction between D.flagrans and infective larvae of nematodes leaded to a new understanding of nematode-trapping process of D.flagrans.By analysis,the nematode-trapping process can be divided into the earlier stage of capture(0 h),the middle of capture(12 h)and the later stage of capture(48 h).The stage division of nematode-trapping process had provided theoretical foundation for the sample collection for transcrip to me research of D.flagrans.(4)The transcript of different nematode-trapping stages were obtained and analyzed based on the reference genome of D.flagrans.The result showed that as many as 41.2%genes were differently expressed during the process of trapping nematodes including 2,877 up-regulated genes and 3,269 down-regulated genes.After GO annotation,some genes related to energy metabolism and transmembrane transport were highly expressed during nematode-trapping process.These genes might be involved in the secretion of extracellaluar proteases and energy production.The KEGG annotation revealed the number of genes related to biosynthesis of secondary metabolite was the most,indicating secondary metabolite may play important roles in nematodes infection.In down-regulated genes,the number of genes related to starch and sucrose metabolization,protein processing in endoplasmic reticulum,peroxisome and carbon metabolism were the most,which indicated the degradation of carbohydrate was weaken which was involved in saprophytic ability of D.flagrans.The analysis of expression of secreted enzymes and the first 100 most highly-expessed genes after extraction of nematodes challenged for 12 h showed that some secreted enzymes related genes were up-regulated such as genes related to lectin,chitinase and subtilisin,while some carbohydrate-degrading related were found down-regulated during nematode trapping process.(5)Based on the selected genes involved in nematode trapping by transcriptome analysis,22 differently expressed genes which were significant to nematode trapping were picked out and validated by real-time fluorescence quantitative PCR.The result indicated both the 11 up-regulated genes and 11 down-regulated genes were the same expressional tendencies with these in transcriptome analysis,indicating the high veracity of transcrip to me data.(6)In order to obtain the protoplast of D.flagrans fast and efficiently to identify gene function,the protoplasts of D.flagrans produced by enzymolysis were labeled with CFDA SE.The optimal dosage and timing as well as the temperature of protoplast labeling were discussed,respectively.The ideal conditions of protoplast labeling were as follows,the final dosage of CFDA SE is 10 μ mol/L,and labeling time is 15 min,with 36℃ as the optimal temperature.The method could provide a new idea for rapid evaluation of the protoplast-producing efficiency from nematode-trapping fungus-D.flagrans. |
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