Establishment Of CRISPR/Cas9 Gene Editing System For Orbilia Oliospora And Study On The Function Of Arthrosporols Biosynthesis Gene

Orbilia oligospora is the most widely distributed and systematically studied nematode predatory fungi.Gene function studies are carried out by gene knockout through homologous recombination,which lacks effective gene editing tool.A class of heterozygous arthrosporols,PKS-TPS,was isolated from the Orbilia oligospora at the early stage of the laboratory,and its biosynthetic gene cluster was identified.However,the biological function of oligosporin and the function of biosynthetic genes are still unclear.Under laboratory conditions after continuous culture,previous build Arthrosporols biosynthesis gene AOL?s00215g283(hereinafter referred to as gene 283)gene knock out strain ?283 spore production capacity loss and mycelial growth slowed,but the specific mechanism is still unclear.Located in arthrosporols biosynthesis gene cluster of another gene AOL?s00215g275(hereinafter referred to as the 275,knockout strains of ?275,expression strains of OE-275)previous studies have shown that was not involved in arthrosporols element synthesis,but the specific function is unknown.In order to solve these problems,this paper carries out researches as following.Key results and innovations:1.Aiming at global regulatory transcription factor 648 gene,CRISPR/Cas9 gene editing system were successfully built in the Orbilia oligospora.The results show that compared with WT,? 648 spore population decline,traps number increase,osmotic stress resistance drop,and ? 648 have new peak.648 in less spore section from spore indicates that the gene not only participate in the regulation of biosynthesis gene cluster but also regulate the spore trap generation and osmotic stress resistance.2.The CRISPR/Cas9 gene editing technique was used to perform point mutation of key amino acids in the predicted binding region on 283,and the optimal conditions for knockout and point mutation were determined in Orbilia oligospora.Metmetabolic analysis showed that 283 had no regulatory effect.In addition,four previously reported active sites(Arg17,Arg18,His33,His34)were found.3.To rebuild 283 mutant strain(?283-E),6-MSA,m-cresol and toluquinol feeding experiments were applied.Metabolic analysis showed ?283-E feeding different concentration of 6-MSA,m-cresol and toluquinol can produce Arthrosporols and lack of different levels of recovery of compound.The results show that 283 does not have regulation effect,but the small molecular compound Arthrosporols has a regulatory role.4.Continuous culture experiment of wild strains WT and knockout mutant strain ?283showed that in three medium PDA,TYGA,YMA,the strain WT and ?283's diameter of the colony,spores,and traps number decreaseed with the increase of the number of batches.The spore producing ability of WT was lost first under the same condition which showed that after gene 283 knock out more conducive to less spore section from spore keep spore production capacity in the process of wedding.?283 and ?283-E for genome analysis,heavy screening to eight can lead to ?283 spore production capacity loss,mycelia growth to slow down,provides the basis for further study ?283 batches mechanism.5.The spore germination rate,hyphae length and different culture medium on catching nematicides rate and quantity analysis of WT and ?283 under the condition of different temperature and different initial concentration showed that WT and ?283 hyphae length increases with the increased concentration of spores,concentration dependence,and after24 h,three temperature hyphae length of WT were not less than ?283,three temperature on WA culture medium,WT and ?283 fishing production and nematicides rate are increased with the increase of concentration of spores,presenting the concentration dependence.And?283 all can in the concentration of spores are lower than those of WT produced apparatus,under 33?.?283 under different concentration of spores trap quantity are higher than that of WT.Three temperature on CMA medium,WT and ?283 fishing production and nematicides rate has not been present concentration dependence.In the condition of 33?,WT and generate ?283 has no nematicides.The results showed that gene 283 was involved in the quorum sensing of Orbilia oligospora.6.For ?275 and OE-275 phenotype,metabolic,transcription,proteomics and metabonomics analysis phenotype analysis showed that 275 regulation of spores oxidative stress resistance and mycelial morphology in the Orbilia oligospora.Transcriptome analysis showed that gene 275 regulates transcription,carbon,nitrogen and energy metabolism.Metabonomics analysis showed that gene 275 was mainly involved in the regulation of arthrosporols and other secondary metabolic synthesis in the Orbilia oligospora.