Role Of H₂O₂ And Function Analysis Of CAT In The Sclerotia Formation Of Rhizoctonia Solani

Maize sheath blight is a worldwide soil-borne disease.With the expansion of the domestic corn planting area,the disease has gradually increased,which has a serious impact on corn production.The pathogenic fungus of maize sheath blight was Rhizoctonia solani.The pathogenic bacteria escaped the adversity in the soil and diseased bodies with the formation of sclerotia and re-emerged under the appropriate conditions.Therefore,it is crucial to the formation and development mechanism of sclerotia,From the analysis of the transcriptome library of the R.solani sclerotia formed by the laboratory,it was found that catalase gene?CAT?has obvious expression differences in the sclerotia formation process,the CAT gene sequence was screened and cloned from the transcription library,and bioinformatics analysis of the amino acid sequence encoded by this gene,study of the role of oxygen stress in the formation of sclerotia by H₂O₂ and CAT interactions,The main results are as follows:1.The primers was designed to clone the full-length c DNA and bioinformatics analysis of its amino acid sequence.The full-length gene is 2014 bp and the sequence is a complete open reading frame,the protein consists of 679 amino acids.The estimated molecular weight was 76.869 KD and the theoretical isoelectric point was 8.56,domain analysis the protein was a typical monofunctional heme catalase,with neither transmembrane structure nor signal peptide,with hydrophilicity,subcellular localization of major functional sites on the mitochondria,phylogenetic analysis showed that CAT protein of Rhizoctonia solani and Laccaria bicolor were the closest to CAT.2.To test effect of different degrees of oxygen stress on the formation of scleoria,H₂O₂ and catalase inhibitors?3-amino-1,2,4-triazo Ie,AT?were exogenously added in the plates of R.solani.The results showed that the concentration of 10-6 mol/L H₂O₂ could significantly promote the growth of mycelia and the development of sclerotia.The concentration of 0.5 mmol/L AT could significantly promote the differentiation of sclerotia,and significantly inhibit the growth rate of mycelia and the development of sclerotia.It indicated that different degrees of oxygen stress had different effects on the growth of mycelia and the differentiation of sclerotia.3.The H₂O₂ concentration and CAT activity in 10-6 mol/L H₂O₂ treatment group and 0.5 mmol/L AT treatment group were detected,the results showed that the intracellular concentration of H₂O₂ in mycelia was 49 ?mol/g,which promoted the growth of mycelia and the development of sclerotia.The intracellular concentration of H₂O₂ in the mycelia of 80 ?mol/g promoted the differentiation of sclerotia.The results of the detection of mycelial CAT activity showed that the CAT activity was positively correlated with the H₂O₂ content,and 0.5 mmol/L AT reduced the CAT activity by approximately 50%.4.The expression of CAT gene relative to sclerotia in R.solani was studied by real-time fluorescence quantitative PCR.The results of RT-q PCR showed that the expression of CAT was very low during the growth stage of mycelium,and it began to rise during the initial stage of sclerotial differentiation,while the expression reached the highest value with maturity of sclerotia,then began to decline.The higher the expression of CAT gene is corresponding to the higher concentration of H₂O₂ in a certain range.The results showed the use of inhibitors did not inhibit the expression of the CAT gene.It is indicated that the CAT gene regulated its own transcription level corresponding to H₂O₂ concentration in the sclerotia to ensure the growth of mycelia and the normal development of sclerotia.