| The diseases in plants are a common kind of various biotic stress and do a damage to the growth and development of plants seriously.The maize,as an important global cereals and industrial materials,various diseases have become one of the vital factors reducing its yield and quality.Therefore,it makes great significance to explore and analyze the resistantrelated genes in maize.According to the previous study,which found antagonistic endophytic Bacillus subtilis DZSY21 can enhance the disease resistance in maize and the analysis of whole genome bisulfite sequencing(WGBS)screened a resistant-related gene,named ZmGRM4.In this study,many works were doing about it.The main results are as follows:1.Bioinformatics analysis found that the ZmGRM4 gene is located on chromosome 5,the full-length of coding sequence of ZmGRM4 is 1905 bp,which encoding 634 amino acids.The molecular weight of its protein is 70.53 k Da and isoelectric point is 7.07,meanwhile it contains typical conserved domain of ethylene receptor protein,such as three transmembrane domains,GAF,His KA.The analysis of phylogeny reconstruction uncovered that ZmGRM4 belongs to the first subfamily of ethylene receptor proteins and its evolutionary progress is conservative relatively.2.Analysis of tissue expression patterns revealed that ZmGRM4 gene is expressed in different tissues of maize,but the expression level was higher in stems,leaves and silks.Analysis of the induced expression patterns found that ZmGRM4 is induced by Curvularia lunata,Pantoea stewartii and Fumonisin B1,meanwhile a slight up-regulated of it induced by SA and JA.Subcellular localization analysis revealed that ZmGRM4 protein is mainly expressed in the cytomembrane.3.Obtaining ZmGRM4 over-expressing transgenic Arabidopsis thaliana.Analysis of the treatment of Pst DC3000 in ZmGRM4 transgenic plant found that ZmGRM4 transgenic plant can increase resistance to Pst DC3000;the disease index,the number of bacterial cells in leaves and the extent of damage in leaves were decreased compared with wild type plants.4.Detecting the expression level of marker genes in SA,JA and ET-mediated signaling pathway found that PR1 and PR2 increased significantly in transgenic plants by Quantitative Real-time PCR infected Pst DC3000 after 24 h.PR1 as a marker gene of SAmediated signaling pathway,Determining SA contant in Arabidopsis thaliana,the results showed that the accumulation of SA in transgenic plants was significantly higher than that in WT;Analysis of the Arabidopsis thaliana pre-treatment by ABT,a biosynthetic inhibitor of SA,found that ZmGRM4 transgenic plants lost resistance to Pst DC3000.The results indicated that ZmGRM4 transgenic Arabidopsis thaliana improve resistance to Pst DC3000 by SA-mediated signaling pathway.5.The p CUB-ZmGRM4-GFP vector was transiently expressed in maize protoplasts,ZmGRM4 gene was up-regulated about 40-fold and the expression levels of SA-related marker gene PR1 increased 8-fold induced by flg22 in maize,which further indicated that ZmGRM4 was involved in plant defense process through SA signaling pathway. |
