Function Analysis Of OsWTF1 And OsWTF2 Promoters In Rice

Rice OsWTF1 and OsWTF2 genes belong to the ERF subfamily of the AP2/ERF family. AP2/ERF transcription factors are involved in the plant-abiotic stress responses, such as drought, cold, salinity, pathogen responses. In this paper, the promoters of the two genes and their different 5'end deletion fragments were fused with GUS gene, and the GUS gene expression patterns were analyzed, in order to reveal the functions of the two promoters. The main results are as follows:1. The sequences of the two promoters obtained from Genebank database were used for promoter analysis using the online PLACE program. The result showed that there are a lot of potential cis-acting elements which are involved in tissue-specific and environmental signaling. Mesophyll-specific element CACTFTPPCA1(YACT), root-specific element ROOTMOTIF(ATATT), pollen-specific element POLL-EN1LELAT52(AGAAA), endosperm-specific element AACACOREOSGLUB1 (AACAAAC), ABRE, MYB, MYC, WRKY, ERF binding sites were found in each of the two promoters.2. The full length OsWTF1 promoter (2049bp) and its different 5'end deletion fragments (1631,608,474,415 bp, respectively) were fused with GUS gene in p1301 plasmid to construct the plant expression vectors. The full length OsWTF2 promoter (2439bp) and its different 5'end deletion fragments (1764,1190,724 bp, respectively) were also fused with GUS gene in p1301 plasmid. All of the constructed vectors were transformed into rice calli by agrobacterium-mediated method.3. The results of histochemical GUS staining showed that the 2049 bp and 1631 bp upstream regions of OsWTF1 gene directed GUS expression in leaves, leaf sheaths, stems, hulls, anthers, roots, seeds and calli. The GUS gene directed by 608 bp,474 bp, and 415 bp upstream fragments expressed in stems, hulls, anthers, roots, seeds and calli. But the GUS activity was not detected in leaf sheaths, and also weak in leaves. The OsWTF2 promoter and 1764 bp,1190 bp,724 bp upstream regions of OsWTF2 gene directed GUS expression in leaves, leaf sheaths, stems, hulls, anthers, roots, seeds and calli. This indicated that the core sequences of rice OsWTF1 gene promoter may be located in the region between-415 and-1 bp, and ten CACTFTPPCA1 elements in the region between-1631 and-608 bp may play an important role in leaf-specific expression. The core sequences of rice OsWTF2 gene promoter may be located in the region between-724 and -1bp.4. Transgenic rice seedlings were treated by different abiotic stresses such as drought, cold, salinity and UV irradiation. The GUS activities directed by pOsWTFl and pOsWTFl-1631 were increased when treated by UV irradiation and salinity. Increased GUS activities directed by OsWTF1 promoter and each of the 5'-deletions were detected when treated by cold and PEG. It can be inferred from the result that, a DRE and two ABRE cis elements, four GT-1 and three WRKY binding sites in the region between-1631 and-608 bp may participate in the induction by abiotic stresses, and there may be some enhancers in the region between-2049 and-1631 bp of the OsWTF1 promoter. On the other hand, the GUS activities directed by OsWTF2 promoter and each of the 5'-deletions increased when treated by drought, cold, salinity and UV irradiation. Interestingly, pOsWTF2-724, pOsWTF2-1764 and pOsWTF2 responded to each abiotic stress more quickly, but pOsWTF2-1190 responded very slowly. We also found that pOsWTF2-724 responded to UV and PEG treatments more dramatically than pOsWTF2-1764 and pOsWTF2. This may indicate that one GT-1, four MYB and three MYC binding sites, one DRE and two ABRE cis elements in the region between-724 and-1 bp may participate in the induction by abiotic stresses, there could be some negative regulatory elements between-1190 bp and-724 bp site of the OsWTF2 promoter, and positive regulatory elements may be located in the region between-1764 and-1190 bp.