Gene Cloning And Expression Of Erebps In Apple

The EREBPs (Ethylene Responsive Element Binding Proteins) are known to be the unique transcriptional factor in plants. The members of this family contain one conserved ERF domains, which consist of about60amino acids. They can specifically bind with plant cis-acting elements GCC box and help to regulate ethylene responses by regulating genes transcription and expression. Two new genes, MdERF-4and MdERF-5, were isolated from an apple cDNA library. The pro-karyotic expression vector PET-32a-MdERF-4(MdERF-5) was constructed and both of MdERF-4and MdERF-5proteins were obtained. The eukaryotic expression vector pPICZB-MdERF-4(MdERF-5) was constructed and identified correctly by PCR, restriction analysis and sequencing. They transformed into Pichia pastoris strains of X-33and KM71H by electroporation and the transformants were obtained by antibiotic selection of the high concentration. But the specific bands didn’t appear in SDS-PAGE and Western blotting analysis about yeast total protein now. The MdERF-4encodes a putative protein containing233amino acid with a predicted molecular mass of26.26KD and contains a conserved ERF DNA binding domain(142to202). The MdERF-5encodes a putative protein containing319amino acid with a predicted molecular mass of35.28KD and contains a conserved ERF DNA binding domain(154to214). The prediction result of protein secondary and tertiary structure and homology analysis suggest that the MdERF-4and MdERF-5are the ERF transcription factors which can activate the expression of GCC-box localized in the nuclear.