Abstract
The promoter activity of chloroplastic carbonic anhydrase gene (ptca1) in the marine diatom Phaeodactylum tricornutum is repressed mediated by cAMP under high CO2 condition. The ptca1 promoter includes CO2/cAMP responsive elements (CCRE1-3), but the details of CO2 signal transduction and regulatory mechanisms of ptca1 promoter is unclear. In this study, we retrieved 8 candidates which may bind to CCREs (PtbZIP1-8) according to the structure of putative DNA binding domain from P. tricornutum genome database. PtbZIP1-8 were cloned and expressed in E. coli, and solubilized PtbZIPs were purified by introduced His-Tag using Ni sepharose. Gel-shift assay was carried out using DNA probes labeled by Cy5; i.e., CCREs pentamers and the native promoter sequence including CCRE1 or CCRE2. The results clearly indicated that PtbZIP7 binds to CCREs specifically, while PtbZIP1 and PtbZIP2 showed non-specific bindings to CCREs. Expression profiles of PtbZIP7 were also analyzed using qPCR, and the localization of PtbZIP7 was observed in P. tricornutum. As a result, transcription levels of PtbZIP7 were constitutive and PtbZIP7 was localized in the nucleus, independently of CO2 concentration.
