| Nomuraea rileyi is an important entomopathogenic fungus which infects lepidopterous pests,in particular Noctuidae spp.,and it has potential in insect biocontrol.The sporulation of N.rileyi requires specific conditions,including light stimulation,and a maltose carbon source.To solve this problem,microsclerotia(MS)of N.rileyi have been induced by liquid amended media(AM)successfully and used as a substitute for conidia in insect biocontrol.The transcriptome and whole genome sequence of N.rileyi have recently become available.These data provide a basis for research on N.rileyi gene function.However,low frequencies of detected HR events are achieved in filamentous fungi using this method because the introduction of integrated DNA into the host genome results in a high frequency of ectopic integration via the non-homologous end-joining(NHEJ)pathway.Therefore,it is necessary to development a high efficient gene disruption method for gene functional research in N.rileyi.In this dissertation,we developed a high efficient gene disruption method of N.rileyi,study the gene functions of Nr Cat1 and Nr Cat2 and the role of these two genes during MS formation by gene knockout.We also study the gene functions of Nr Pex16 by gene knock.The findings of this research will be of great importance in reveal the role of ROS metabolism during MS development,and will facilitate to guide the liquid fermentation of MS in N.rileyi.Main results of this study are as follows:(1)Development of high efficient gene disruption method by ATMT1)Construction of split-marker vectorsThe split-marker vectors p PZP-Split-marker-A and p PZP-Split-marker-B were derived from p PZP-Hph-Knockout by enzyme digest and ligase.We also insert a gus cassettes in p PZP-Split-marker-B as a negative selection marker,the new plasmid was named p PZP-Split-marker-B-gus.2)Efficiency of ATMT and gene disruptions efficient of split-markerThe transformation efficiency of linear cassettes method was 11 times higher than split-marker.Disruption Nr Cat1,Nr Cat2 and Nr Pex16 in using linear cassettes and splitmarker method,we found that the gene disruption efficiency of linear cassettes were 1.6%,4.2% and 1.0% respectively and the gene disruption efficiency of split-marker were 81.0%,82.3% and 64.1% after negative selection.(2)The research on catalase genes Nr Cat1 and Nr Cat4 in N.rileyi1)Cloning and sequence analysis of Nr Cat1 and Nr Cat4Two catalases ORF sequences were cloned and designated as Nr Cat1 and Nr Cat4 respectively.The ORF sequence of Nr Cat1 is 2651 bp,encoding 716 amino acid residues.The ORF sequence of Nr Cat4 is 1130 bp,encoding 385 amino acid residues.Pridiction of conserved domains we found that the proteins encode by Nr Cat1 and Nr Cat4 belong to catalase-like super family and both these two proteins have a heme binding domain.2)The roles of the catalase genes Nr Cat1 and Nr Cat4 play in MS formationCombined with mutant phenotype analyses we found that ?Nr Cat1 was unable to form MS structure,the fermentation broth had a paler pigment compared with WT,the MS formation also delayed,in the anaphasis of MS formation.?Nr Cat4 was able to form MS structure and the pigment of fermentation broth was darker than WT,the MS formation was in advanced than WT.Combined stage-specific expression pattern analysis of Nr Cat1 and Nr Cat4 genes,we found that Nr Cat1 and Nr Cat4 were involved in ROS regulation and metabolism during MS formation.These results indicated that both Nr Cat1 and Nr Cat4 are involved in oxidative stress during MS formation,delete these two genes will impact the formation of MS.3)The roles of the catalase genes Nr Cat1 and Nr Cat4 play in vegetative growth,conidiation,germination and stress resistance.Germination,development,sporulation,and stress resistance analysis of WT and mutant strains have shown that the hypha extension rate was 1.1 times than WT,the sporulation quantity was as 82% as WT and the germination time of mutant was advanced than WT.These results indicated that deletion of Nr Cat1 impact the vegetative growth,conidiation,germination.H2O2 assay indicated that deletion of Nr Cat1 will decrease the resistant ability of H2O2,indicated that Nr Cat1 was involved in exogenous H2O2 stress.Deletion of Nr Cat1 and Nr Cat4 decrease the thermotolerance of N.rileyi indicated that Nr Cat1 and Nr Cat4 are involved in heart stress.?Nr Cat4 and?Nr Cat4 are insensitive with high osmotic pressure,and?Nr Cat1 sensitive with congo red.4)Nr Cat1 and Nr Cat4 is required for full virulence against S.litura hostIn topical infection assay,both ?Nr Cat1 and?Nr Cat4 mutants showed less virulence in S.litura than WT.Combined with the catalase activity assay,we found that the lower catalase activity the lower virulence.(3)The research on peroxin Nr Pex16 in N.rileyi1)Cloning and sequence analysis of Nr Pex16The ORF sequence of Nr Pex16 is 1157 bp,encoding 385 amino acid residues.Pridiction of conserved domains we found that the proteins which encode by Nr Pex16 belong to Pex16-like super family.2)The roles of the peroxin Nr Pex16 play in vegetative growth,conidiation,germination and stress resistanceThe loss of Nr Pex16 leads to the deficiency of vegetative growth and conidiation.Compared with WT,?Nr Pex16 showed a much slower switching from yeast-cell to hyphae on SMAY and the deletion mutant decrease the germination rate of conidium.Stress resistance assay has indicated that?Nr Pex16 is sensitive to oxidative stress,heat stress and congo red,but insensitive to high osmotic pressure.3)The role of Nr Pex16 in fatty acid metabolism in N.rileyi?Nr Pex16 is able to grow on MM medium which use maltase and Na Ac as carbon source,but unable to grow on MM which use triglyceride phosphate,olive oil and Tween-80 as carbon source.4)Nr Pex16 is required for full virulence against S.litura hostIn topical infection assay,?Nr Pex16 mutants showed less virulence in S.litura than WT.The reason of this phenotype may due to the poor growth,decreased ROS metabolism and unable to metabolism the fatty acid which in the host.Conclusion: In this dissertation we developed a high-efficiency gene disruption method of N.rileyi,the efficiency of this method is higher than 80%.Nr Cat1 and Nr Cat4 are catalase genes,mutant anyone of these two gene will lead the compensatory of the other one.We also found that Nr Cat1 and Nr Cat4 are related to stress resistant,virulence,spore germination and formation of MS,the Nr Cat1 also impact the growth of hyphal extension and sporulation.In N.rileyi delection of Nr Pex16 will impact the hyphal growth,sporulation,spore germination,stress resistant,virulence,fatty acid metabolism. |
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