| Arthrobotrys oligospora can produce a special class of Arthrosporols which can regulate the morphology of Arthrobotrys.Our group previously identified 9 key genes 283-276 and 274,which locate in AOL s00215g gene cluster and are involved in the biosynthesis of oligosporins.However,the biosynthetic functions of these five genes,274,280,276,277 and 278,have not been completely determined.Therefore,this study will exploit the functions of these genes by constructing mutants with double knock-outs and analyzing the chemical profiles.Key results and innovations:1.Using hygromycin and chlorimuron-ethyl as screening markers,four mutants with double knockouts of Arthrobotrys oligospora were successfully constructed.The four double-knockout mutant strains were ?280A274,?259A911,?278?277,?276?277.2.The mycelium morphology of the double-knockout strain A280A274 was more similar to that of the single-knockout strain A280,and more luxuriant than that of the single-knockout strain ?274.The growth rate of strain A280A274 was 28.0%,30.3%and 30.7%slower than that of strain A280,A274 and wild strains respectively.In 24h,the number of traps of A280A274 was 0.2 times,2.5 times and 5.7 times higher than that of strain ?280,?274 and wild strains respectively.In spore yield,A280A274 was 98.9%less than wild type and 99.4%less than A274,but twice as much as A280.The effects of gene 280 knock-out on mycelial morphology,spore yield and trap number were greater than that of gene 274 knock-out,so ?280?274 on mycelial morphology,spore yield and trap number was closer to that of A2803.The mycelial morphology of double-knockout strain ?259?911 was more similar to that of single-knockout strain A259,but its growth rate was 6.7%faster than that of single-knockout strain A259,7.3%slower than that of single-knockout strain?911 and 17.2%slower than that of wild strain.The spore yield of ?259?911 was 81.2%lower than that of ?259,and more than 99%lower than that of ?911 and wild strains.Gene 259 knock-out had more influence on mycelial morphology,spore yield and trap number than gene 911 knock-out,and the mycelial morphology,spore yield and trap number of ?259?911 were closer to ?259.4.The mycelium morphology of double-knockout strain ?278?277 was more similar to that of single-knockout strain ?278,but its growth rate was 5.0%faster than that of single-gene knockout strain ?278,5.6%slower than that of single-gene knockout strain ?277 and 7.3%slower than that of wild-type.The strain A278A277 was 57.4%less than 278,and 99%less than A277 and wild strains.Knockout gene 278 had more influence on mycelial morphology and spore yield than knockout gene 277,and?278?277 was more s,imilar to ?278.5.In PDA,TYGA and YMA media,the growth rate of double-knockout strain?276?277 was 7.8%slower than that of single gene knockout strain ?276.In PDA and TYGA media,the growth rate of ?276?277 was 2.93%faster than that of single-knockout strain ?276.At 12h,the number of traps of A276A277 was increased 30%higher than ?277 and 4.3%higher than A276,while the wild type had no mature three-dimensional network.The spore yield of ?276?277 was 16.0%lower than that of?276,30.4%lower than that of wild strain,but 17.7%higher than that of ?277.The deletion of genes 276 and 277 had different effects on mycelial growth,mycelial morphology and spore yield,and the mycelial morphology was more similar to the wild type.6.The peak of[M+H]+ was 343.226310 when the retention time was 35.98 min,which corresponded to 343.22632 of epoxide,the key intermediate of oligosporin biosynthesis.At the same time,the characteristic peaks of ?280?274 and ?274 were 275.163[M+H]+(19.34 min),313.20059[M+H]+(20.67 min).343.22638[M+H]+(35.92 min),327.23126[M+H]+(39.47 min),respectively.There are two identical characteristic peaks with ?280:243.08742[M+H]+(15.80 min)and 267.11878[M+H]+(18.38 min).After gene 259 was knocked out,the metabolic map changed greatly,and a lot of products disappeared,which was similar to the change after gene 283 was knocked out,and the metabolic map of ?911 was the same as that of wild type.The metabolic map of the double knock-out strain ?259?911 was identical to that of ?259.The metabolic map of the double knockout strain ?277?278 was consistent with that of?277,and there were no heterozygous small molecules containing C15 in the metabolic map of ?277,but there were heterozygous small molecules containing C15 in ?278.This indicates that gene 277 acts in front of gene 278.The metabolic map of double knock-out strain ?276?277 was consistent with ?276,and there were no PKS-TPS heterozygous small molecules in the metabolic map of A276,and the loss of peak was more than ?277.The results showed that the role of gene 276 was prior to gene 277 in the pathway of oligosporin biosynthesisIn this study,double-knockout strains were constructed to study the effects of two genes on mycelial growth,mycelial morphology,spore yield,spore germination.,trap number and nematode-trapping ability.It was found that the single-knockout strains were more likely to cause greater variation in morphology,spore germination,trap number and nematode-trapping ability. |
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