Functional Analysis of the HbREF1 Promoter from Hevea brasiliensis and Its Response to Phytohormones

The rubber elongation factor (REF) is the most abundant protein in the latex of Hevea brasiliensis, which is closely related to natural rubber biosynthesis. In order to gain a deeper understanding of the transcriptional regulation mechanism of HbREF1, a 1758 bp genomic DNA fragment of the HbREF1 promoter was isolated. Promoter sequence analysis revealed several transcription factor binding sites in the HbREF1 promoter, such as bZIP, bHLH, EIL, AP2/ERF, MYB, and Trihelix. To assess the promoter activity, a series of HbREF1 promoter deletion derivatives were created and fused with firefly luciferase (LUC). The LUC image demonstrated that all of the HbREF1 promoters exhibited transcriptional activity. Furthermore, the assay revealed the presence of multiple regulatory elements within the promoter region that negatively regulate the transcriptional activity. Subsequent analysis of the transcriptional activity following treatment with phytohormones identified an ABA-responsive element located between -583 bp and -200 bp, an ET-responsive element between -718 bp and -583 bp, a JA-responsive element between -1758 bp and -1300 bp, and a SA-responsive element between -1300 bp and -718 bp. These results were largely consistent with the predictions of cis-acting elements. This study has established significant groundwork for future investigations into the regulatory mechanism of HbREF1.