Cloning And Functional Analysis Of Three ERF Genes In Melon

ERF(ethylene responsive factor)is a downstream transcription factor of the ethylene signal transduction pathway that can regulate a series of biological processes including stress response,plant growth and fruit ripening.ERF transcription factors belong to the EREBP subfamily of the AP2/EREBP superfamily and contain an AP2/ERF binding domain.Several ERF transcription factors have been cloned in melon already,but there are few reports on its function of melon fruit ripening to date.Melon(Cucumis melo L.)is a plant of family Cucurbitaceae cucumis with nutrient-rich fruit.Therefore,it is necessary to study the role of ERF transcription factors played in melon fruit ripening.In our study,CmERF?-12,CmERF?-14 and CmERF?-2,genes were cloned according to the sequences acquired from the melon database and they encode 275,148,391 amino acids respectively.The physicochemical properties of the proteins,phosphorylation site,signal peptide structure,transmembrane structure and other related properties were analyzed by bioinformatics software.The result revealed that all CmERF?-12,CmERF?-14 and CmERF ?-2 transcription factors are non-secretory hydrophilic proteins,possessing different phosphorylation sites.These three proteins do not belong to membrane proteins as they lack transmembrane structures.The subcellular localization prediction showed that CmERFV-2 and CmERF?-14 locate in the nucleus while CmERF?-12 mostly distributes in the cytoplasm.Therefore,the three transcription factors may play different regulatory roles in the nucleus and cytoplasm.To study its primary function,RNAi interference and overexpression vectors of CmERF?-12,CmERF ?-14 and CmERFV-2 were constructed.An agrobacterium-mediated transformation of melon was used for transient expression.Real-time quantitative PCR was applied to detect the expression characteristics of three transcription factor genes and the relative expression level of fruit ripening-related genes in transient transformation sites.The fruit peel showed an early-emerging yellow color in the injection site of CmERF ?-14 gene overexpression vector and remained green when RNAi was transformed into them.The fruit peel color was green after the introduction of CmERF?-2 gene overexpression vector and yellow after RNAi introduction.Neither overexpression nor RNAi treatment of CmERF?-12 gene changed the phenotype of peel color significantly.The result according to qPCR revealed that overexpression of CmERF?-14 increased the expression level of ripening related genes ACS4,EXP1 and PSY1,while the interference treatment reduced the expression level.Besides,overexpression of CmERF?-2 reduced the expression level of gene EXP1.Therefore,CmERF?-2 may delay the maturation of melon fruit while CmERF?-14 accelerate it.CmERF?-12 may has no or little effect on melon fruit ripening.In addition,the reporter vector pPZP-E8-GUS carrying the promoter of fruit-specific E8 gene was constructed.The reporter vector was co-transformed into tobacco by leaf injection with overexpression vectors(pPZP-ERF?12,pPZP-ERF?14,pPZP-ERF?2)acting as effect vector.According to the change of GUS activity,CmERF?12 and CmERF?-14 gene may activate fruit-specific E8 promoter and accelerate gene expression to increase the relative activity of GUS enzyme.However,CmERF?-2 may reduce the relative activity of the enzyme by suppressing the activity of E8 promoter.