Study On The Functions Of Three Biosynthesis Related Genes In Arthrobotrys Oligospora And Construction Of Thermophilic Fungus Thermomyces Lanuginosus 200065 Mutants

Fungi produce abundant secondary metabolites with significantly biological activity.The unique arthrosporols compounds produced by Arthrobotrys oligospora play roles in the growth,sporulation and predation of the fungus.Thermophilic fungus Thermomyces lanuginosus 200065 yielded analogues of the nematocidal compounds thermolides.However,the biosynthetic pathways of these compounds remained largely unknown.In this study,we first identified the functions of three genes AOL_s00215g294,AOL_s00173g162 and AOL_s00080g313.The genetic engneering for construction of T.lanuginosus 200065 mutants were also performed.Threes genes AOL_s00215g294,AOL_s00173g162 and AOL_s00080g313 were selected as the target genes.The knockout vectors of three genes were constructed by in-Fusion method.CaCl2-PEG mediated protoplast transformation was applied to yield transformants.The mutant strains of three genes were screened and confirmed by PCR.These mutants showed significant differences in morphology,mycelial growth,sporulation,spore germination rate,trap formation and nematode predating ability.?AOL_s00080g313 showed fast mycelial growth and spore germination and much more traps than the wild-type strain.Within 6 h,mutant ?AOL_s00080g313 could develop many trap formations with the number at 452/cm2,while the wild-type did not produce traps yet.After 12 h,the number of traps of ?AOL_s00080g313 reached to 895/cm2 but that of wild-type strain was only 14/cm2.However,?AOL_s00080g313 showed significantly decreased sporulation by 99.8%compared to the wild-type strain.The other two mutants?AOL_s00215g294 and ?AOL_s00173g162 also displayed similar decreases in sporulation HPLC and GC-MS analysis revealed that the three mutants display significantly increased peaks compared with the wild-type strain.A unique compound benzeneacetic acid detected in the mutant ?AOL_s00080g313,together with homovanillyl alcohol detected in the wild-type strain suggested that gene AOL_s00080g313 might be involved in hydroxylate C-4 in phenylacetic acid and its analogues.The biosynthetic genes in the whole genome of T.lanuginosus 200065 were predicted and analyzed in this thesis.Seven biosynthetic genes were selected for the targets,including Thela2₀05217_t1,Thela2₀01896_t1,Thela2₀03207_t1,Thela2₀05753_t1,Thela2₀00273_t1,Thela2₀02 779_t1,Thela2₀05333_t1.The Agrobacterium tumefaciens-mediated transformation were applied for the genetic engineering.However,highly efficient non-homologous recombination in the T.lanuginosus led to all the ectopic transformants.To reduce ectopic insertion,methods to improve homologous recombination need to be further explored,such as CRISPER-Cas9 to study on the functions of thermophilic fungi biosynthetic genes.The functions of three biosynthetic genes in A.oligospora were studied.The disruption of the gene AOL_s00080g313 caused the most significant differece in fungal phenotype and metabolic pattern.The transformation method of Agrobacterium tumefaciens-mediated T.lanuginosus 200065 was applied to explore the genetic engineering for the thermophilic fungus.