Cloning And Functional Analysis Of The HbERF From Hevea Brasiliensis

Ethylene responsive element binding protein ERF is a subfamily of AP2/ERFEBP transcription factor family. The transcription factor has a conserved AP2/EREBP domain consists of60amino acids. In the life history of plants, ERF plays an important role in the regulation of the growth and development, cell differentiation and responsing to a variety of hormones and stresses. Based on the full-length sequences of seven ERF-encoding genes from the latex of Havea brasiliensis, in this study, we cloned the cDNA and DNA sequences of seven genes, and made an intensive study about structure feature, expression characteristics, prokaryotic expression, biochemical characteristics, and protein interaction of several genes. The main results are as follows:1. The cDNA and DNA full-length sequences of HbERFl-7were cloned. Sequences analysis shows that, cDNA sequences of HbERF1/2,5-7were consistent with DNA sequences, indicating that the five genes contained no introns. The cDNA full-length of HbERFS and HbERF4were777bp and1158bp while their DNA sequences were918bp and2627bp. The analysis result shows that each of HbERF3and HbERF4contained an intron, and the length was respectively141bp and1472bp.Structure analysis shows that HbERF1-7contain typical AP2domains. The homology between HbERFl-7and some higher plants such as Ricinus communis, Populus trichocarpa, Vitis vim/era. Solarium lycopersicum are between49%and87%. We classified HbERFl, HbERF2and HbERF6in cluster B2, HbERF2in cluster B4, HbERF3and HbERF4cluster B2and HbERF7in cluster B6by cluster analysis with18ERF members from Arabidopsis.2. The1572bp5’upstream promoter region of Hb ERF3gene was cloned and sequence analysis showed that the fragment contains typical eukaryotic core promoters and a transcription start site. The analysis of the cis-acting elements shows that the fragment contains a variety of elements such as that response to hormone and stresses, such as ABRE, ARR1AT, CGTCA-motif MYCCONSENSUSAT, WRKY71OS and so on, which provides evidence to the research of ERF.3. We analyzed the expression of HbERFl-5in different tissues of Havea brasiliensis, and in leaves under the treatment of different hormones (JA, SA, ABA, ET). The result shows that, the expression of HbERFl, HbERF3and HbERF4were highest in leaves, while the expression of HbERF3in latex was consistent with that in leaves. HbERF2was highest in bark, HbERF5was highest in root. Under the treatment of JA and SA, the expression of most genes showed a trend of first increasing and then decreasing, while under the treatment of ABA and ET, the expression was elevated to high expression amount and remained unchanged.4. Seven prokaryotic expression vectors pGEX-HbERF1-7were constructed and HbERF1/2/5/7were expressed in E.coli successfully. Three eukaryotic over-expression vectors pCAMBIA1304-HbERFY5/6/7were constructed and HbERF7was transformed into tobacco. Fifteen transgenic plants were obtained and physiological detection and functional identification related is on-going. 5. Five yeast two-hybrid system bait vectors were constructed and proved have no transcriptional activation activity so that they can be used in yeast two-hybrid experiment. By yeast two-hybrid technique, the proteins interacting with HbERF5were screened, and rubber elongation factor (REF), a protein abundant in latex, and a subunit of AP2domain were successfully screened.