Application of the fluorescent protein mCherry to study the inducible promoter rd29A under low temperature conditions in Arabidopsis

Abstract In this study, we investigated the application of mCherry, a fluorescent protein, to evaluate the inducible promoter rd29A in Arabidopsis under low temperature, using the Arabidopsis genome as a template to amplify this promoter by PCR. Overlapping extension PCR was used to ligate the rd29A promoter and the mCherry gene to initiate the expression of mCherry through construction of a pHDE-rd29A-mCherry plasmid. Competent Agrobacterium cells were transformed with the recombinant plasmid, and Agrobacterium-mediated transfect Arabidopsis to obtain the progeny which was identified containing rd29A-mCherry in the transformed plants. Positive transgenic T1 seeds were planted in 1/2MS medium, and the seedlings were subsequently cultured at 34, 24, 14, 4 and 0°C for 20 h. Expression of the mCherry gene was then determined using an inverted fluorescence microscope. The results showed that fluorescence intensity was significantly higher in seedlings grown at 4°C compared with those grown at other temperatures. Furthermore, the rd29A promoter was induced to express low-levels of mCherry expression. Quantitative PCR assay indicated that mCherry expression was high under the 4°C condition, which was consistent with the results obtained by microscopy. These findings indicate that mCherry can be used as an exogenous marker to investigate spatio-temporal promoter activation.