Studies Of Ascorbate Peroxidase In Response To Resistance To Southern Corn Leaf Blight

Maize is the first largest food crop in China and plays an important role in ensuring our food security.However,in recent years,with the global warming and the change of farming and planting system,maize diseases have shown a more and more serious trend in production,seriously reducing the yield and quality of maize.Southern corn leaf blight（SCLB）is one of the most important leaf diseases in maize production.It occurs widely in the world and brings severe challenge to maize production.It is an important way to solve this problem to explore SCLB resistance resource,analyze its resistance mechanism,and cultivate resistant varieties.We previously identified a gene ascorbate peroxidase located in the interval of QTL for resistance to SCLB,that encoded ascorbate peroxidase,and the results indicated that ascorbate peroxidase was associated with resistance to SCLB.On the basis of previous studies,the function of ascorbate peroxidase on resistance to SCLB in maize was analyzed by analyzing the expression of ascorbate peroxidase in disease resistant and susceptible inbred lines.The mutant materials of ascorbate peroxidase were further screened and identified,and the resistance of the mutant lines was identified,which confirmed the important function of ascorbate peroxidase on the resistance of SCLB.Finally,the resistance mechanism of ascorbate peroxidase on SCLB was analyzed by cytology and molecular biology.The main research results are as follows:1.The expression of ascorbate peroxidase in disease resistant inbred line Mo17 and disease susceptible inbred line B73 was determined by qRT-PCR.The results showed that there was no significant difference in the expression level of ascorbate peroxidase between Mo17 and B73 without the inoculation of Bipolaris maydis.However,the expression level of ascorbate peroxidase in Mo17 was significantly higher than that in B73 after the inoculation of Bipolaris maydis.The above results suggested a positive regulatory role of ascorbate peroxidase on the resistance of maize to SCLB.2.Two Mu transposon insertion materials,mu1043915 and mu1046680,were planted in the field.The homozygous mutant lines of ascorbate peroxidase was screened and identified by PCR,and the relative expression level of ascorbate peroxidase gene and ascorbate peroxidase protein in the homozygous mutant lines and wild type lines were further studied by qRT-PCR and Western blot.The results showed that the expression level of ascorbate peroxidase gene in the homozygous mutant lines was significantly lower than that of the wild type lines,and the expression level of ascorbate peroxidase protein was also significantly lower than that of the wild type lines,indicating that the ascorbate peroxidase mutant lines identified by screening could be used for subsequent experiments.3.The resistance of ascorbate peroxidase homozygous mutant lines and wild type lines to SCLB was further identified.The results showed that the resistance of the homozygous mutant lines to SCLB was significantly lower than that of the wild type lines.The cytological differences between the homozygous mutant lines and the wild type lines after inoculation with Bipolaris maydis were studied by paraffin section method.The results showed that the number of mycelia in the homozygous mutant lines was significantly more than that of the wild type lines,and the degree of cell damage was significantly more serious than that of the wild type lines.The results further demonstrated the function of ascorbate peroxidase on resistance to SCLB.4.The accumulation of H₂O₂ in ascorbate peroxidase homozygous mutant lines and wild type lines after inoculation with Bipolaris maydis was determined by DAB staining and H₂O₂ detection kit.The results showed that H₂O₂ content in the homozygous mutant lines was higher than that of the corresponding wild type lines at different sampling time points after inoculation.The H₂O₂ content of Mo17 and B73 inbred lines inoculated with Bipolaris maydis were further determined.It was found that the H₂O₂ content of Mo17 was significantly lower than that of B73.These results suggested that ascorbate peroxidase may enhance maize resistance to SCLB by reducing H₂O₂ level.5.Finally,the accumulation of defense-related hormones JA and SA in ascorbate peroxidase homozygous mutant lines and wild type lines after inoculation with Bipolaris maydis was measured by ELISA kit.The results showed that the JA content of the homozygous mutant lines was significantly lower than that of the wild type lines after inoculation with Bipolaris maydis.The SA content of the homozygous mutant lines was not significantly different from that of the wild type lines,indicating that ascorbate peroxidase did not affect the change of SA content in maize,but could promote the accumulation of JA.The expression levels of JA and SA synthesis and response related genes were further determined by qRT-PCR.The results showed that the expression levels of JA synthesis and response related genes Zm AOS,Zm LOX2,Zm OPR8,Zm PDF1.2 and Zm VSP2 in homozygous mutant lines were significantly lower than those of the wild type lines.The expression levels of SA response related genes Zm PR1 and Zm PR5 were not significantly different from those of the wild type lines.These results indicated that ascorbate peroxidase may regulate maize resistance to SCLB through JA mediated signaling pathway.In summary,the ascorbate peroxidase homozygous mutant lines were successfully identified in this study,and the function of ascorbate peroxidase on resistance to SCLB was further studied by using the mutant lines.It was found that ascorbate peroxidase could reduce the accumulation of H₂O₂,increase JA content and participate in JA signaling pathway to enhance maize resistance to SCLB.These results clarified the function and preliminary resistance mechanism of ascorbate peroxidase in SCLB resistance,broadened the research of ascorbate peroxidase in plant disease resistance,and provided a novel resistance resource for maize resistance breeding.