Cloning And Functional Analysis Of MdARF3 Gene In Apple

ARF3(auxin response factor 3)is a plant transcription factor that responds to auxin and plays an important role in the transduction pathway of auxin signal.The ARF3 gene has been studied in Arabidopsis,but rarely in apple.In the earlier period,homologous tetraploids were obtained by doubling the homologous chromosomes of the ?Hanfu? apple,and it was found that the resistance of the autotetraploid was higher than ?Hanfu? apple.Transcriptome sequencing results showed that the ARF3 gene expression was significantly higher in the ?Hanfu? apple than its autotetraploid.This experiment cloned the ARF3 gene coding region sequence from ?Hanfu? apple.On the one hand,the prokaryotic expression vector was constructed by MdARF3 gene,and the induced MdARF3 protein was used to screen the interacting proteins.On the other hand,an overexpression vector of MdARF3 gene was constructed.Transgenic apple plants were obtained by genetic transformation,which laid the foundation for the study of the functional genes in apple.The main results of this experiment are as follows:1.The full-length cDNA of MdARF3 gene was cloned from ?Hanfu? apple.The full-length cDNA of MdARF3 was 2136 bp and contained a complete reading frame,encoding a total of 712 amino acids.It was homologous to ARF3 in other species.The analysis found that the ?Hanfu? apple ARF3 protein contains two conserved functional domains in the ARF gene family: the DNA-binding domain(DBD)and the middle domain(MR).Through the phylogenetic analysis of ARF3 in other plants,it was found that the ARF3 gene has an evolutionary relationship between plants.2.The relative expression level of MdARF3 gene in fruits was the highest,followed by the shoot tips,and the relative expression levels in flowers,roots and leaves were lower.3.The subcellular localization showed that the Md ARF3 protein was localized in the nucleus.4.The GST Pull-down assay was used to screen the Mal d 1 protein interacting with Md ARF3 protein,and the interaction between MdARF3 and Mal d 1 protein was verified by yeast two-hybrid.5.Using Agrobacterium tumefaciens-mediated leaf disctransformation method,the ?Hanfu? apple MdARF3 gene was introduced into ?GL-3?apple and 4 transgenic plants were obtained.6.The inoculation of M.aeruginosa infestation revealed that the transgenic apple plants overexpressing the MdARF3 gene were more susceptible to disease.The results showed that the MdARF3 leads to reduce the disease resistance of plant.