Study On The Function Of Catalase Gene Of Curvularia Lunata

Catalase is one of the key enzymes to regulate the balance of active oxygen in organisms,which can decompose hydrogen peroxide into water and oxygen.Hydrogen peroxide exists in organisms as a form of active oxygen and can be used as a signal molecule at low concentrations.Excessive concentration of hydrogen peroxide can cause oxidative stress,which can seriously lead to cell death.Curvularia leaf spot is a corn leaf disease caused by Curvularia lunata(Walk)Boed.,but its pathogenesis is not very clear.Therefore,the catalase gene of corn Curvularia leaf spot CX-3 strain is taken as the research object in this paper and five catalase genes knocked out mutant are obtained to clarify the function of catalase gene and the regulation mechanism of related signaling pathways,analyze the influence of this gene on the growth and pathogenic ability of pathogens,and lay the foundation for further study on the pathogenic mechanism of C.lunata.The results are as follows:1.Obtaining the complete catalase gene sequence and bioinformatics analysis of Curvularia leaf spot.According to the known Cl CAT1,Cl CAT2,Cl CAT3,Cl CAT4,and Cl CATlike genes in C.lunata m118,the entire sequences of Cl CAT1,Cl CAT2,Cl CAT3,Cl CAT4,and Cl CATlike in C.lunata CX-3 were obtained by Blast alignment.Using online analysis software analysis,the Cl CAT1 gene contains seven exons,the sequence length is1117 bp,encodes 333 amino acids,and the protein is located in the cytoplasm.The evolutionary tree analysis was found to have the highest homology with powdery mildew.The Cl CAT2 gene is 2501 bp in length,encodes 736 amino acids,has 21 exons,and is mainly distributed extracellularly.The analysis of the phylogenetic tree revealed that it has the highest homology with CAT3 of Heterosporus.The Cl CAT3 gene sequence is 2723 bp in length,encoding 740 amino acids,and the number of exons is 21;it is mainly distributed on the peroxisome;it has the highest homology to CAT2 of Heterosporus.Cl CAT4 gene sequence is 1833 bp in length,encoding 571 amino acids,and the number of exons is 10;it is mainly distributed on peroxisome and cytoplasm,and has the highest homology with Candida albicans.The Cl CATlike gene sequence is 1257 bp in length,encoding 404 amino acids,andthe number of exons is 10;it is mainly distributed in the cytoplasm,and is also distributed in peroxisomes and mitochondria,and has the highest homology with Candida albicans.2.The knockout vectors p PZP100HGCAT1,p XEHCl CAT2,p XEHCl CAT3,p XEHCl CAT4,p XEHCl CATlike were successfully constructed and introduced into Agrobacterium Agl-1.Agrobacterium tumefaciens-mediated genetic transformation(ATMT)was used to obtain ? Cl CAT1,? Cl CAT1-C,? Cl CAT3,and ? Cl CAT4.3.Biological experiments show that Cl CAT1,Cl CAT3 and Cl CAT4 have significant effects on the early spore germination,biomass,intracellular antioxidant capacity,pathogenicity against external oxidative stress,osmotic stress and pathogenicity of Curvularia lunata.?Cl CAT4 is sensitive to external oxidative stress,which indicates that Cl CAT4 gene regulates the ability of corn Curvularia leaf spot to resist external oxidative stress.?Cl CAT1 is sensitive to Na Cl.This indicated that the Cl CAT1 gene affected the ability of maize Curvularia leaf spot to resist osmotic stress.4.Analysis of the regulation of related signal pathway genes by Cl CAT1,Cl CAT3,Cl CAT4 genes of Curvularia leaf spot bacterium in corn.In order to fully understand the regulation of CATalase gene on signal pathway of corn C.lunata,this paper analyzed the expression of key genes of signal pathway related to the influence of catalase gene by q RT-PCR technology.it was found that cat gene of corn C.lunata had regulation and close correlation on g protein signal pathway,MAPK protein kinase pathway and light signal pathway genes.Among them:(1)G protein signaling pathway,Cl CAT1 gene has negative regulation on nop-1 gene and Cl CAT3 gene has positive regulation on nop-1 gene.Cl CAT3 and Cl CAT4 have positive regulation on gna-1 gene.The Cl CAT1 gene has negative regulation on nop-1 gene,and the Cl CAT3 gene has positive regulation on nop-1 gene.Cl CAT3 and Cl CAT4 play positive regulatory roles on gna-1 gene.(2)MAPK protein kinase pathway,Cl CAT1 gene has negative regulation on div-4,stk-38 and apg-1 genes,and positive regulation on stk-4 genes.Cl CAT3 and Cl CAT4 have positive regulation on div-4,stk-38,apg-1 and stk-4 genes.(3)light signal pathway,Cl CAT1 gene has negative regulation on wc-1and wc-2 genes.Cl CAT3 and Cl CAT4 genes have positive regulation on wc-1 and wc-2 genes.