Isolation And Molecular Characterization Of AP2/EREBP-like Protein From Rice, Oryza Sativa L.

Plant growth, development and crop yield are severely influenced by numerous abiotic and biotic stresses. However, plants also evolved a series of complex response and resistance mechanism during the long term interaction with these adverse environmental conditions. There are many reports suggested that plant AP2/EREBP superfamily transcription factors play important functions in many plant developmental processes and in response to environmental stresses. Therefore, to study the AP2/EREBP type proteins is significantly of importance to understand the mechanism of plant response to stress conditions.From previous constructed SSH cDNA library of two different groups, one of which was ABA treated rice seed embryos and another was normal germinated rice seed embryos (Wuhan university, Professor Zhao Jie's Lab). The dot-blotting method was employed to screen and identify the differentially expressed clones, a novel AP2/ EREBP-like protein gene encoding putative ethylene responsive element binding factor (ERF)/AP2 domain was isolated by screening of the ABA-treated SSH cDNA library from rice plant and designated as OsAP2LP. Its full length of 1491bp was obtained by RACE-PCR and sequenced, from which a 348 amino acid residues protein was deduced with a predicted weight of 39.1kDa. A phylogenetic analysis revealed that OsAP2LP contains a highly conserved AP2 domain.Semi-quantitative RT-PCR analysis revealed that OsAP2LP gene was highly expressed in root, stem, 30DAP (day after pollination) embryo and 2 days germinated seed embryo. In addition, expression of OsAP2LP gene was induced by ZT (zeatin), ABA (abscisic acid), high salinity (NaCl) and dehydration, whereas it did not respond to JA (methyl jasmonate) and SA (salicylic acid).Promoter sequence analysis indicates that the upstream region of OsAP2LP gene possesses some elements induced by physiological and environmental factors. These results suggest that OsAP2LP might be involved in the process of plant response to diverse abiotic stresses through an ABA-dependent way and might have a role in the cytokinin signaling pathway. And T-DNA insertion of OsAP2LP caused lethal phenotype to rice plant when the seedlings were at 3-week-old stage. This suggest that this novel AP2/EREBP gene in rice might play non-redundant function, as a transcription factor, loss of function mutation might fail to effectively transcrible its corresponding downstream target genes, thereby leading the seedlings to death.In this study, another AP2/ERF type transcription factor-OsTSH1 isolated from the SSH cDNA library, the cloning and expression pattern analysis of which have been finished by former researcher. To further investigate the biological function in plant, over expression of rice OsTSH1 gene in Arabidopsis was performed. In all the positive transformants, OsTSH1 effectively induced the activation of various of stress-responsive genes, such as RD29A, PR1, PDF1.2. These results suggest that OsTSH1 may be involved in process of plant response to both abiotic and biotic stresses and enhance the plant tolerance by activating corresponding downstream stress-responsive genes.