Study On The Role Of P5cr Gene In Synthesis Of Swainsonine In Metarhizium Anisopliae

Locoweed is one of the main poisonous weeds that endanger livestock husbandry.Animal feeding on locoweed shows poisoning symptoms characterized by chronic neurological disorders.swainsonine(SW),the toxic substance of locoweed,is produced by its endophytic fungus Alternaria oxytropis.However,the study of its swainsonine synthesis pathway has been seriously hampered due to its slow growth rate and difficulties in protoplasmic preparation.Meanwhile,Metarhizium anisopliae,as an important pest biocontrol agent,is also one of the fungi producing swainsonine,and some studies have been carried out on the synthetic pathway of swainsonine organisms in M.anisopliae.However,studies on the role of pyrroine-5-carboxylate reductase(p5cr)in the swainsonine biosynthetic pathway have not been reported.In this study,we used Metarhizium anisopliae(Metschn.)Sorokin as a target,and performed functional validation of the catalase gene p5 cr after knockdown and overexpression by CRISPR/Cas9 gene knockdown and overexpression techniques.The aim of this study was to verify the regulatory role of p5 cr in an important step of the biosynthetic pathway(from lysine(Lys)to Pipecolinic acid(PA)synthesis)of swainsonine in M.anisopliae(Metschn.)Sorokin,as well as to validate the application of CRISP/Cas9 gene editing technology in M.anisopliae(Metschn.)Sorokin.To lay the theoretical foundation for the study of the biosynthetic pathway and related catalase genes of swainsonine.The following results were obtained in the study:1.There is a strong correlation between the yield of swainsonine and the expression of p5 cr geneThe level of swainsonine in M.anisopliae(Metschn.)Sorokin fermentation broth and mycelium on day 1-7 was detected by UPLC-MS/MS.The expression of p5 cr and other related genes(sdh,p450 and pks)in the synthesis pathway of M.anisopliae was measured by q RT-PCR.Using bivariate correlation analysis,it was found that the level of swainsonine was strongly correlated with the expression of pks gene(r=0.800)and highly correlated with the expression of sdh,p5 cr,and p450 genes(r=0.820,0.860,and 0.820,respectively)in 7days of fermentation in M.anisopliae(Metschn.)Sorokin.2.Establishment of p5 cr deletion strains and overexpression strains of Metarhizium anisopliaeFor the optimal conditions of M.anisopliae(Metschn.)Sorokin.protoplast preparation,it was found that the maximum concentration and the best state of protoplasts were obtained when the enzymatic combination was 1% RWX(1% snailase + 1% cellulase + 1%lysozyme)and the enzymatic time was 6 h.The p5 cr deletion strain(Δp5cr)was obtained by transferring CRISPR/Cas9 knockout vector and homologous donor DNA into wild-type(W)strain protoplasts to knock out the p5 cr gene using PEG8000-mediated methods.The p5 cr overexpression vector PBARGPE1-p5 cr was transferred into the protoplasts of the strain using the same method to obtain the p5 cr gene overexpression strain(PBARGPE1-p5cr).3.the effect of overexpression and deletion of the p5 cr gene on the phenotype of the strainThe colony morphology of M.anisopliae(Metschn.)Sorokin W strain,PBARGPE1-p5 cr and Δp5cr strains were observed,and the cellular microstructure was observed using upright microscope and transmission electron microscopy.It was found that the Δp5cr strain showed angular change,and the color of PBARGPE1-p5 cr and Δp5cr colonies was more yellowish than that of W strain,while no other obvious changes were found in the fine structure.The growth rates of M.anisopliae(Metschn.)Sorokin W,PBARGPE1-p5 cr and Δp5cr strains were further measured on a medium containing different concentrations of Congo red(CR).The results showed that,compared with the W and Δp5cr strains.It was observed that the growth rate of overexpression strain PBARGPE1-p5 cr was not significantly inhibited with the increase of Congo red concentration compared with W and Δp5cr strains,suggesting that overexpression of p5 cr gene enhanced the barrier effect of cell wall to some extent.4.the effect of overexpression and deletion of the p5 cr gene on swainsonine contentThe swainsonine level of W,PBARGPE1-p5 cr and Δp5cr strains was measured by UPLC-MS/MS,and the expression level of p5 cr gene as well as sdh,p450 and pks genes in the swainsonine pathway was measured by q RT-PCR.The results showed that the swainsonine level of PBARGPE1-p5 cr strain was increased by 4.4-fold and the swainsonine level of Δp5cr was significantly decreased(p<0.01)compared with the W strain.The lysine level of PBARGPE1-p5 cr strain was decreased(p<0.05)and the lysine level of Δp5cr strain was increased(p<0.05)compared with the W strain.Pipecolinic acid level was increased in PBARGPE1-p5 cr strain(p<0.01)and decreased in Δp5cr strain(p<0.01)compared with the W strain.There was no change in sdh and pks gene expression in W,PBARGPE1-p5 cr andΔp5cr strains,while the p450 gene expression was significantly increased(p<0.05)in the overexpressing strain PBARGPE1-p5 cr compared with the W strain.The results confirmed the critical role of p5 cr in the swainsonine synthesis pathway from lysine to Pipecolinic acid synthesis in M.anisopliae(Metschn.)Sorokin.However,the pathway in which p5 cr is located may not be the only pathway or there may be isozymes that are present.5.Effect of overexpression and deletion of p5 cr gene on the expression of pathogenic genes of Metarhizium anisopliaeQRT-PCR was used to detect the expression levels of M.anisopliae pathogenic genes(including chitinase chit42,catalase cat1,and M.anisopliae adhesin-like protein mad1 and mad2 genes)in W,PBARGPE1-p5 cr and Δp5cr strains to predict fungal pathogenicity changes.The results showed that the virulence genes detected in the p BARGPE1-p5 cr andΔp5cr strains did not significantly change comparing to the wild strains,which verified that the p5 cr gene affected the level of swainsonine while not significantly affecting the virulence genes of M.anisopliae(Metschn.)Sorokin.In conclusion,we have successfully obtained CRISPR/Cas9-HDR(homologous recombination repair)mediated p5 cr gene deletion strains of M.anisopliae(Metschn.)Sorokin and obtained p5 cr overexpression strains in this study.It was verified that the p5 cr gene plays an important role in the regulation of swainsonine biosynthesis pathway in M.anisopliae.Furthermore,the p5 cr gene in Metarhizium has a high affinity(100%)to the p5 cr gene of A.oxytropis.Therefore,the results of this study will lay an important theoretical foundation for the subsequent exploration of the swainsonine biosynthetic pathway and catalasegenes of swainsonine.