Cloning And Functional Analysis Of ERF Transeription Factor Genes Related To Low Temperature In Rubber Tree(Hevea Brasiliensis)

Cold-resistant breeding is one of the important objectives in rubber tree breeding. Because of the long breeding cycle of rubber tree and the lack of early prediction means, it is useful to improve the cold resistance of rubber tree by modern molecular biological techniques, so the process of cold-resistance breeding can be accelerated in rubber tree. In this study, we obtained partial cDNA contained conservative ERF sequences from full-length cDNA library, and cloned HbERF1by RACE. The expression of HbERF1was analyzed by real-time fluorescence PCR under cold stress in rubber tree. To investigate the function of HbERF1, it was overexpressed in Arabidopsis thaliana. All of the results give a base of cold-resistant breeding in rubber tree. The detailed results are as follows.ERF is a kind of stress-resistant transcription factors, which can activate or inhibit the expression of defense genes, and play a crucial role in signal transduction between different signaling pathways. We successfully cloned the HbERFl by RACE in rubber trees. The full length sequence of HbERF1was1178bp containing a639bp ORF, and encoded213amino acid. The deduced protein was23.43kD with an isoelectric point of8.96.Bioinformatic analysis of the HbERFl gene shows that:HbERF1has one conservative AP2domain belonging to the AP2/ERF family members and can regulate the gene expressions under abiotic stress like other plant ERF genes. The deduced amino acid sequences contained no signal peptide, transmembrane domain and obvious hydrophobic region.The expressions of HbERF1in different tissues and under cold stress were assayed by real time fluorescent PCR. The results showed that the tissue-specific expressions of HbERF1were flowers> root> bark> leaves; the expression of HbERF1not only was up-regulated under cold stress but also cumulative increase with the increase of stress time.In order to investigate its function under abiotic stress, HbERF1was cloned into the expression vector35S promoter-drived pXCS-HAStrep and overexpressed in Arabidopsis thaliana. Fifteen transgenic lines of Arabidopsis thaliana were obtained, of which, Southern blotting results showed that line S1and line S3were single copy insertion and double copy insertion, respectively. qPCR results showed that HbERF1was high expression in both transgenic lines, and the expression of line S1was significantly higher than that of line S3.Overexpression of HbERF1inhibit the of ethylene response genes expression and enhanced the salt resistance ability in Arabidopsis.COR15a,a low temperature-related gene, were significantly up-regulated in two transgenic lines, and enhance the cold resistance of Arabidopsis thaliana. The expression analysis of RD22and RD29A were carried out in wild type Arabidopsis and transgenic lines, respectively. The results showed that the expressions of the two genes were very low under normal conditions in wild type and transgenic Arabidopsis, and the expression patterns under drought and high salt stress tended to consistency. It is indicated that there is an complex signaling pathway when HbERF1regulates the downstream genes.