Identification And Functional Analysis Of The Interaction Protein MdbHLH92 Of Powdery Mildew Disease Resistance Gene MdERF100 In Apple

Apple(Malus domestica)is one of the most widely cultivated fruit tree species because of its flavor and nutritional value.However,powdery mildew is one of the most common and harmful diseases in the life cycle of apple,which seriously restricts the yield and quality of apple.In the early stage,our research group analyzed and screened out a differentially expressed gene MdERF100 in response to powdery mildew stress from transcriptome level,and its function of powdery mildew resistance was preliminarily proved by heterologous transformation of Arabidopsis thaliana.On this basis,MdbHLH92 was found by yeast two-hybrid technology and bimolecular fluorescence complementation technique(Bi FC),which was a interaction protein of MdERF100.The regulatory role of MdbHLH92 in powdery mildew stress was explored in Arabidopsis thaliana,which providing a new idea for improving the regulatory network of powdery mildew in apple and a new basis for molecular breeding of disease resistance.The main results are as follows:1.Expression analysis of MdERF100 gene in ‘Gala’ showed that MdERF100 was expressed in roots,stems,leaves,flowers and young fruits,and its expression level was high in leaves and roots.The expression level of MdERF100 gene was significantly upregulated under exogenous SA,JA and ethylene treatments,confirming that the gene could respond to multiple hormone treatments.Gala-3 was genetically transformed by the leaf disc method ediated by agrobacterium,which laid a foundation for the subsequent functional study of MdERF100 gene.2.The interaction protein MdbHLH92 of MdERF100 was found by yeast two-hybrid technique,and the interaction between MdbHLH92 and MdERF100 was verified by Bi FC.The total length of MdbHLH92 transcription factor cloned from ‘Gala’ was 708 bp,encoding235 amino acids,including a HLH conserved domain.Subcellular localization experiments found out that MdbHLH92 protein was localized on cell membrane.Yeast self-activation test made clear that MdbHLH92 don’t have transcriptional self-activation activity.A 1536 bp promoter fragment from the upstream of MdbHLH92 was cloned and found to contain multiple cis-acting elements related to stress.3.The expression analysis of MdbHLH92 showed that MdbHLH92 was induced by powdery mildew and could respond to exogenous SA,JA and ethylene hormones.In addition,tissue-specific analysis made clear that MdbHLH92 was expressed in roots,stems,leaves,flowers and young fruits,and the highest expression level was found in leaves.4.The MdbHLH92 overexpression vector was constructed and heterologous transformation of Arabidopsis thaliana was performed to obtain T3 positive lines.After inoculation with powdery mildew,phenotypic observation,spore count statistics and histochemical staining were carried out 7 days after inoculation.The results showed that,compared with wild type Arabidopsis thaliana,the disease was lighter,the number of spores decreased significantly,the number of dead cells and the accumulation of reactive oxygen species were significantly higher.Meanwhile,it was discovered that the spore germination,mycelium growth and secondary sporophyte production of transgenic plants were inhibited by comparing the growth and development of powdery mildew.By analyzing the expression of disease resistance genes,it was speculated that overexpressing MdbHLH92 could enhance the resistance to powdery mildew by participating in SA and JA signal transduction pathway.