A Preliminary Study On The Function Of Non-ribosomal Peptide Synthetase 4(ClNPS4) Of Curvularia Lunata

Maize Curvalaria Leaf Spot,which caused by Curvularia lunata,is one of the major leaf diseases in maize and threats the yield of maize.Research on C.lunata mainly focus on cell wall degrading enzymes,non-host selective toxins,melanin and nutrition element iron.Little work has been done on the molecular mechanisms of its secondary metabolic pathways and their effects on pathogenicity.Non-ribosomal Peptide Synthetase（NRPS）is responsible for the synthesis of low molecular weight secondary metabolites,in which NPS6 and NPS2 are conserved and functionally stable in multiple filamentous fungi,respectively,and is closely related to the biosynthesis of the extracellular siderophore and intracellular siderophore.Moreover,NPS4 plays an important role in the surface hydrophobicity of fungi and the establishment of conidia morphology.At present,the effect of NRPS on growth and pathogenic mechanism of C.lunata is unclear.In this paper,NPS4 of C.lunata was cloned,phylogenetic tree and its domain was predicted.The knockout vector of ClNPS4 was constructed and the mutant was obtained by ATMT（Agrobacterium tumeficans-mediated transformation）,which analyzed the biological characteristics of ClNPS4.It was of great significance to of the further understanding of the pathogenicity of C.lunata in view of the secondary metabolic,and guiding the prevention and control of this disease.Results were as follows:1.ClNPS4 was cloned and analyzed by bioinformatics method.ClNPS4 was obtained from NCBI and analyzed by bioinformatics.Results showed that the full-length DNA sequence of ClNPS4 was 21606bp（Gen Bank: KY471559.1）,including an open reading frame,and encoded 7201 amino acids.Through systematic phylogenetic tree analysis with other organisms NPS4,it indicated that ClNPS4 had the highest homology with ChNPS4,which phylogenetic relationship was the closest.Domain analysis showed that the domain of ClNPS4 was completely consistent with the ChNPS4,and consisted of 19 modules,which are composed of C,A,T,and E domains,we predict that ClNPS4 may have similar functions with ChNPS4.The chemical formula of the metabolite was predicted to be C₁₀N₄O₄H₁₇,but the product was not purified for structural analysis.2.The ClNPS4 knockout mutant and complementary mutant were obtained by ATMT.Based on the information obtained by bioinformatics analysis,the knockout and complementary vectors were constructed using C.lunata CX-3 as a template by traditional digestion method and overlap PCR combined with one-step cloning method.Vectors were transformed into A.tumefaciens strain AGL-1 by electroporation.The mutant and the complementary strain were obtained by ATMT.The positive mutants were screened by antibiotic selection and verified by PCR to ensure the reliability.3.Effects of NPS4 on the development and pathogenicity of C.lunata.The conidia germination rate test showed that the conidia germination rate of ΔClnps4 was lower than the wild-type and supplementary strain.Biomass test showed that the mycelium dry weight of ΔClnps4 was significantly higher than that of the wild-type and complementary strain.The virulence of ΔClnps4 was reduced comparing with the wild-type strains and supplementary strain.The detection of secondary metabolites showed that ClNPS4 had effect on the synthesis of secondary metabolites.However,there were no significant differences in colony morphology,growth rate,conidia production,stress resistance and hydrophobicity among the knockout mutant,wild-type strain and complementary strain.