Establishment Of Agrobacterium-mediated Transformation System Of Fusarium Solani And Study On Genes Related To Growth

Fusarium spp.is widely distributed and has a wide variety of species.In agriculture,it seriously affects plant growth by infecting plants and producing toxins,which can cause root rot,ear rot,seedling wither and other diseases of various economic crops.They lead to the reduction of crop yields and causes huge economic losses to agricultural production.Clinically,as an opportunistic pathogen,Fusarium spp.can cause superficial infections such as keratitis,onychomycosis,cellulitis,etc.;The incidence of invasive infections caused by Fusarium spp.,such as endocarditis,fungalemia,and pneumonia,has gradually increased in recent years.Among the fungal infections caused by Fusarium spp.,F.solani accounts for the largest proportion,especially causing a high incidence of fungal keratitis,which can cause blindness in severe cases;For immunocompromised populations,F.solani causes invasive infection with high mortality.Due to the multi-drug resistance of Fusarium spp.,there is currently no effective drug for clinical treatment,which causes harm to the health and life of patients.Therefore,it is urgent to analyze the gene function of F.solani and explore its drug resistance mechanism.In this study,43 clinical isolates of Fusarium were identified by morphology and molecular biology,and there were 5 species,among which F.solani was the most(25isolates,accounting for 58.1%).The minimum inhibitory concentrations of several common antifungal drugs(itraconazole,voriconazole,amphotericin B and caspofungin)against Fusarium were determined according to the CLSI liquid-based microdilution method.The results showed that Fusarium was the most sensitive to amphotericin B,voriconazole had better antibacterial effect,itraconazole had poor bacteriostatic effect,but caspofungin had no antibacterial effect.A preliminary understanding of the susceptibility of Fusarium to commonly used antifungal drugs,and provides a basis for clinical treatment.From the above isolates,a drug-resistant F.solani(HD25)was selected as the wild type,and a plasmid containing a geneticin resistance selection tag was used to construct an Agrobacterium-mediated genetic transformation system of F.solani.In order to improve the transformation efficiency,the transformation conditions were optimized.Agrobacterium tumefaciens(OD₆₀₀=0.8)and F.solani spore suspension(1×10⁴CFU/ml)were mixed in equal volume and co-cultured at 22°C for 48h,the transformation rate can reach 170 transformants/10⁴ spores.The system can be used for both random insertion mutation of the genome and targeted knockout of genes,providing a feasible and effective experimental technique for in-depth study of gene function.A total of 213 mutants of F.solani were obtained through the ATMT system established above.The mutants were screened by phenotype,and a total of 3 strains with slowed colony growth were obtained.Among them,FSM156 produced orange pigment.The drug sensitivity of the mutants was screened,and the results showed random The drug sensitivity of the selected mutants did not change.In our previous work,our group discovered a gene related to the growth of F.oxysporum,and there is a homologous gene(named FSOG in this study)in F.solani,which encodes a hypothetical protein,The exact function is not yet clear.Analysis of the domain of the hypothetical protein found that some of its regions were similar to the Zip A domain.In order to further analyze the function of this gene,the FSOG gene was knocked out in the wild type of F.solani by using the established Agrobacterium-mediated genetic transformation system of F.solani,and the knockout strain?FSOG was obtained.Phenotypic observation showed that,compared with the wild type,the knockout strain?FSOG showed slow colony growth and abnormal conidia morphology.The wild-type and knockout strains were inoculated on the medium containing Congo red and SDS,observation of the colony growth,that the knockout strain?FSOG showed tolerance to Congo red,indicating that the deletion of this gene could affect the integrity of the Fusarium cell wall.The expression levels of genes related to the cell wall integrity pathway(CWI)were detected by q PCR.The results showed that the expression levels of slt2,rlm1,fks2 and other genes were down-regulated;at the same time,the expression levels of genes involved in cell wall synthesis were detected,and the results showed that chitin The gene expression level of chitin synthase(CHS)was partially up-regulated.The above results indicate that this gene can affect the composition and integrity of the cell wall by regulating the CWI pathway and the enzymes related to cell wall synthesis.The study found that Zip A is a key protein involved in cell division in Escherichia coli.Other studies have shown that cell wall integrity has a regulatory effect on the cell cycle,so this study further analyzed the expression levels of proteins and kinases involved in cell cycle regulation.The results showed that the expression levels of genes encoding related proteins and kinases in the knockout strain?FSOG were down-regulated,indicating that this could affect the growth of F.solani through regulating the cell cycle.In conclusion,this study successfully established an Agrobacterium-mediated genetic transformation system of F.solani,and applied it to both random insertional mutagenesis and gene-directed knockout.A knockout strain of the F.solani was successfully constructed,and it was found that the gene could regulate cell wall synthesis-related enzymes,CWI pathway-related genes,cell cycle-related proteins and kinases,affecting the growth of F.solani.The results of this study will help to explore the unknown functions of genes,and lay the foundation for the search for disease-related genes and the screening of potential drug targets.