A Preliminary Study On The Function Of Superoxide Dismutase (SOD1) Of Curvularia Lunata

Superoxide dismutase is one of the most important enzymes of organisms that regulate the balance of reactive oxygen metabolism,as well plays a vital role in signal transduction in vivo.Curvularia leaf spot disease caused by Curvularia lunata(Walk)Boed is an important corn leaf disease.Its pathogenesis is still not very clear.In this paper,the superoxide dismutase SOD1(ic Cu/Zn-SOD)of C.lunata was used as the research object.The gene was cloned and knocked out,and the regulation of SOD1 on the regulation of related signal pathways and the growth,pathogenicity and stress of pathogens were analyzed.Clearing the function of the gene and laying the foundation for further enriching the pathogenic mechanism of C.lunata.The detailed methods and results are as follows:1.Identify the basic structure of SOD1 of C.lunata and its physical and chemical properties.According to the known complete sequence of SOD1 gene in C.lunata strain m118,the complete sequence of SOD1 gene in C.lunata strain CX-3 was obtained by Blast alignment.On this basis,the characteristics of SOD1 gene and protein of C.lunata were analyzed.The gene was 1032 bp in length and contained 4 exons,encoding 154 amino acids.SOD1 is localized in the cytoplasm and is a stable hydrophilic protein with no signal peptide.Phylogenetic tree analysis revealed that the SOD1 gene of C.lunata CX-3 strain has the closest phylogenetic relationship with the SOD1 gene of C.lunata strain m118,and the homology is as high as 94%.2.Obtainment of SOD1 gene knockout and complement mutants of C.lunata.In this experiment,the SOD1 gene knockout vector and the complementary vector were constructed and transferred into Agrobacterium tumefaciens AGL-1 by electroporation.The SOD1 knockout and complement mutants were successfully obtained by ATMT technology and the obtained mutants were screened by antibiotics and verified by PCR.3.Analysis of the regulation of related signaling pathway genes by the SOD1 gene of C.lunata.In order to fully understand the regulation of SOD1 gene on the signal pathway of C.lunata,this paper analyzes the key genes related to the signal pathway of SOD1 gene by q RT-PCR.The results indicate that the SOD1 gene acts on the retinoid(nop-1)in the G protein signaling pathway,the serine/threonine-protein kinase ste20(stk-4)in the MAPK protein kinase pathway,and the trihydroxynaphthalene(brn-1)in the melanin synthesis pathway.4.SOD1 gene affects the biological function and pathogenicity of C.lunata.DAB staining showed that after the SOD1 gene was knocked out,the concentration of hydrogen peroxide at the tip of the mature hyphae decreased,indicating that the SOD1 gene is a key gene for scavenging superoxide ions.In the stress-tolerant test,the sensitivity of ?SOD1 to hydrogen peroxide,menaquinone and sodium chloride was significantly higher than that of wild type,indicating that SOD1 gene affects the resistance to oxidative and osmotic stress of C.lunata.At the same time,morphological observation showed that the synthesis of hyphae melanin in ?SOD1 was significantly reduced in the early growth stage,indicating that SOD1 gene has a regulatory effect on melanin synthesis.The inoculation test showed that the SOD1 gene had no significant effect on pathogenicity.