Isolation And Function Analysis Of Grape ERF Genes

Plant growth and productivity are strongly influenced by abiotic stress such as drought, high salinity or low temperature. Under stress conditions, plants firstly sense the environmental changes trough many ways, and then transfer these changes into the cell as signals, and then transcription factors accepted these cell signals will act with cis-acting elements and regulate the expression of stress responsive genes, which will then result in an enhancement of stress-tolerance in plants. Therefore, transcription factors play a pivotal role in stress tolerance of plants.In this research, the plant stress tolerance-related genes were investigated in two ways:1. The sequences containing the AP2/ERF domain were acquired from the TIGR grape gene index database by Bioinformatic methods.Nine ERF genes were analyzed by Bioinformatic methods:1) By the bioinformatic methods and tools, the composition of amino acid sequences, signal P, phosphorylation sites , tertiary structure,genome localization information and functional domains were predicted and analyzed to nine ERF genes. The results demonstrated that all of them are secretory proteins containing signal peptide except ERF2-1, ERF2-2, ERF3c. And these nine genes all have a complete AP2/ERF domain; besides,there are multiple serine phosphorylation sites in every ERF genes.2) An investigation of phylogenetic relationship and the sequence conserved motif of the ERFs in Vitis vinifera were performed and compared with those of Arabidopsis. As a result of the analysis, the AP2/ERF domain was quite conserved in the v.vinifera and Arabidopsis. Grape ERF genes show high homology with Arabidopsis. They are useful for us to study of grape ERF genes. In addition, we isolated five ERF genes VvERF2-2,VvERF3a,VvERF3b,VvERF3c,VvERF4-1 from grape for subsequent researches.2. In order to investigate the functions of ERF genes in V.vinifera further, we chose two ERF genes VvERF3a and VvERF3b from five genes for further analysis. The results as follows:1) We studied on their expression levels induced by high salinity, ET, SA and ABA by using a semiquantitative analysis method. It showed that VvERF3a and VvERF3b were sensitive to ET, SA and ABA, but they didn't response to the stress of high salinity. The results suggested that they may involve in the signal pathways of SA, ET and ABA.2) Transactivation activities of VvERF3a and VvERF3b were determined by a yeast system. The results shows that the yeast cells carrying the fragments of VvERF3a and VvERF3b could grow on SD/-Trp-His-Ade medium, respectively.3) In order to investigate biological functions, overexpression construct of VvERF3a and VvERF3b genes were made and transformed into Arabidopsis by Agrobacterium-mediation method.To sum up, in this study we obtain 9 ERF genes by electronic cloning; five ERF transcription factor genes were isolated from grape (v.vinifera); some molecular charaterizations of VvERF3a and VvERF3b were analyzed and transgenic Arabidopsis overexpressing the VvERF3a and VvERF3b genes were acquired for further research.