Studies On The Functions And Expression Profile Of HbEBP1 Gene From Hevea Brasiliensis

Rubber tree is perennial tropical crop, and long time period is needed for the conventional breeding process. Screening a large-scale promotion improved strains extends to 25-30 years in generally, and the selection rate is very low, only several out of thousands. Moreover, the abiotic stress have seriously affected the yield of rubber tree, especially the cold climate and the drought. It is becoming the breeding goals that using the plant molecular biology technology to identify genes related to some important traits, and combine with the traditional breeding techniques for innovating the rapid growth、cold and drought resistant rubber tree cultivars.In this research, we screened the HbEBP1 (ErbB-3 binding protein 1) gene from rubber tree cDNA library. The nucleotide sequences were analyzed, and functions were predicted by bioinformatical method. Their expression was studied by Northern blotting method. Besides, we overexpressed HbEBPl in Arabidopsis thaliana, and its function was characterized. The main results were summarized as follows:1. The full length sequence of HbEBP1 was 1462 bp, contained 141 bp 5’UTR and 133 bp 3’UTR, and deduced encoding 395 amino acid, which was predicted to be 43.596 kD and with isoelectric point of 6.45.2. Bioinformatics analysis indicated that HbEBP1 had three conserved domains:a proliferation-associated 2G4(PA2G4-like) domain, a metallopeptidase M24(APP_MetAP) domain, a methionine aminopeptidase(MAP) domain. The predicted peptide contained no signal suequence, transmembrane domain or obvious hydrophobic region, suggesting its localization was in the cytoplasm and nucleolus. Other analysis suggested this protein played functions in the process of signal-transduction, and regulated growth and stress-response as well.3. Successfully constructed the prokaryotic expression vector (pGEX-6p-1)-HbEBPl, and expressed in E.coli BL21 (DE3).After 4h induction with 0.1mM IPTG at 37℃, the GST-HbEBP1 fusion protein was obtained. The fusion protein was purified and the MAP activity was assayed. The results showed the prokaryotic expressed HbEBP1 protein had no MAP activity, though a MAP domain was predicted existing in the peptide seueqnece.4. The T-DNA vector pXCS-HbEBP1-eGFP-HAStrep was successful constructed, and transferred into Arabidopsis. The HbEBP1 subcellular localization was identified by observing the eGFP distribution. Strong GFP signal was found in the cytoplasm and nuclear, indicating the HbEBP1 protein located in the cytoplasm and nucleolus as well.5. Northern Blotting results showed that HbEBPl was induced by low temperature, drought and ABA in a similar the express timeline. At the beginning of the stress, the HbEBPl expression increaseed rapidly in 30 min, then decreased to the low level. After 2h, HbEBP1 expression rised again and maintain at certain level.6. Several homologous HbEBP1 overexpressed Arabidopsis thaliana lines were obtained. Northern blot experiments results showed that HbEBP1 expression was detected in these plansts. Southern blot comfirmed single copy insertion in these lines. The transgenic lines phenotype was observed. The seeds of transgenic lines germinated 1-2days earlier and grew more rapid than WT. When grown up, the OE plants had larger rossete and leaf area than that of wild type. Besides, the OE line plants showed stronger root system, with more lateral root than the control.7. In vivo stress treatment experiments revealed of HbEBPl overexpression enhanced the drought tolerance of transgenic plants, but did not influence their cold resistance.