| 摘要 |
The Wcs120 gene encodes a highly abundant protein which appears to play an important role during cold acclimation of wheat. To understand the regulatory mechanism controlling its expression at low temperature (LT-responsiveness), the promoter region has been characterized. To identify the regulatory elements involved in LT-responsiveness, transient expression activity or different promoter regions was determined using the luciferase reporter gene. The data indicate the involvement of putative enhancer elements, negative and positive regulatory regions in the transcriptional regulation of this gene. The role of nuclear factors on the promoter region was analysed. Electrophoretic mobility shift assays using short promoter fragments revealed the presence of multiple DNA-binding proteins in nuclear extracts from non-acclimated (NA) plants which interact with several elements. In contrast, no DNA-binding activity was observed in the nuclear extracts from cold-acclimated (CA) plants. in vitro dephosphorylation of CA nuclear extracts with alkaline phosphatase restored the binding activity. Moreover, okadaic acid (a potent phosphatase inhibitor) stimulated markedly the in vivo accumulation of the WCS120 family of proteins. This suggests that protein phosphatases PP1 and/or PP2A negatively regulate the expression of the Wcs120 gene. In addition, both Ca<2+>-dependent and Ca<2+>-independent kinase activities were found to be significantly higher in the CA nuclear extracts. Western analysis using antibodies directed against protein kinase C (PKC) isoforms showed that a PKCg homolog (84 kDa) is selectively translocated into the nucleus in response to low temperature. Taken together, our results suggest that, in vivo, the Wcs120 gene expression may be regulated by nuclear factors whose binding activity is modulated by a phosphorylation/dephoshorylation mechanism. Further, the promoter was found to be cold-inducible in different freezing tolerant and sensitive monocot and dicot species, suggesting that universal transcription factors responsive to LT may be present in all plants. Therefore this promoter could be used to drive the genes needed for LT tolerance in sensitive species. |
