Cloning And Transcriptional Activity Analysis Of MdERF2 Gene Promoter

Apple is the climacteric fruit,and ethylene plays a vital role in its development.ERF family transcription factors are key factors that directly affect target genes in the ethylene signal pathway.MdERF2 gene is an important transcription factor for ethylene to regulate apple fruit development and quality,but the mechanism and effect of ethylene on MdERF2 are still unclear.In this paper,the characteristics of the MdERF2 gene promoter sequence are analyzed.The transient expression vector containing ?-glucosidase(GUS)and Green fluorescent protein(GFP)is used to double study and verify the regulation mode of MdERF2 gene promoter by ethylene and its inhibitor 1-MCP.The main results are as follows:The MdERF2 gene promoter was amplified by PCR.Sequence analysis showed that the amplified promoter is 1348 bp in length.The Plant CARE and PLACE databases were used to analyze the cis-acting elements contained in the gene promoter.The promoter sequence has basic elements such as the TATA box and the CAAT box,as well as other elements such as GARE-motif,CCAAT-box,Box 4,GT1-motif and MBS.pRI-ProMdERF2-GUS promoter transient expression vector was consructed,and the agrobacterium-mediated method was used to transfect tomato fruits and leaves transiently to verify the active of the promoter.The results showed that: GUS gene driven by Pro MdERF2,under three treatments of 1-MCP,ethephon and blank,tomato fruits and leaves are blue,and the blue color is lighter than that in positive control.The blue under the three treatment conditions is not different significantly,which indicating that Pro MdERF2 has transcriptional activation activity,which is not as active as Ca MV 35 S promoter.pRI-ProMdERF2-GFP transient expression vector was constructed,and the PEG induction method was used to transiently transfect tobacco protoplasts to verify the active of the promoter.The results showed that under the three treatments of 1-MCP,ethephon and blank,the GFP gene driven by Pro MdERF2 has a fluorescent signal in the tobacco protoplasts,but the fluorescence signal under the three treatment conditions is not significantly different.The results also showd that Pro MdERF2 has transcriptional activation activity and has the basic function of a promoter.