The amplified coding region of JcMADS40 without the termination codon was inserted into this vector (pSAT6-eYFP-N1) to generate 35S::JcMADS40-YFP. The 35S::JcMADS40-YFP fusion expression construct and the 35S::YFP empty vector were transferred into Arabidopsis protoplasts using the polyethylene glycol-mediated method. Subcellular localization of the control YFP and JcMADS40-YFP fusion proteins was observed under a confocal laser scanning microscope. Arabidopsis protoplasts were obtained following Tang [5 (link)].
For the transactivation assay of JcMADS40, the full-length JcMADS40 gene was fused to the pBD vector to generate the construct pBD-JcMADS40. This construct and the p5 × GAL-Reporter vector were introduced into Arabidopsis protoplasts. A ProteoPrep® Total Extraction Sample Kit (Sigma) was employed to isolate total protein of Arabidopsis protoplasts according to the operating instructions of the kit, then the fluorescence activity of proteins was analyzed using the enzyme-labeled instrument. The LUC/REN ratio was used to measure the transcriptional activation of JcMADS40.
For the transactivation assay of JcMADS40, the full-length JcMADS40 gene was fused to the pBD vector to generate the construct pBD-JcMADS40. This construct and the p5 × GAL-Reporter vector were introduced into Arabidopsis protoplasts. A ProteoPrep® Total Extraction Sample Kit (Sigma) was employed to isolate total protein of Arabidopsis protoplasts according to the operating instructions of the kit, then the fluorescence activity of proteins was analyzed using the enzyme-labeled instrument. The LUC/REN ratio was used to measure the transcriptional activation of JcMADS40.
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