Fluorescence flx800 microplate fluorescence reader

The full-length SlbHLH59 ORF was cloned into the effector vector pGreen II 62-SK under the control of CaMV 35S promoter. SlPMM, SlGMP2 and SlGMP3 promoter fragments were PCR amplified using specific primers and cloned into the reporter vector pGreen II 0800-LUC. Individual combinations of effector and reporter vectors were co-transformed into Agrobacterium GV3101 cells alongside the pSoup vector, and the transformed GV3101 cells were used to infiltrate young N. benthamiana leaves, in which transient expression was analyzed following a 2-d incubation. Firefly and Renilla luciferase signals were assayed with the dual luciferase assay reagents (Promega) using an Infinite M200 (Tecan).
The promoter activity analysis was carried out as described previously [62 (link)]. GUS activity and LUC activity were determined by Fluorescence FLx800 microplate fluorescence reader (BIO-TEK Instruments). Ratios of GUS to LUC activities were used to define relative promoter activity. Three biological replicates were performed for each construct. The cis-element analysis was conducted in PLACE (http://www.dna.affrc.go.jp/PLACE).

For promoter activity analysis, a series of mutated pCTB4a::GUS fusion constructs were used for transient transformation into Arabidopsis protoplasts. The CaMV35S::LUC plasmid was used as an internal transformation control to provide an estimate of the extent of transient expression. The co-transferred protoplasts were incubated for 16 h at 25 °C and 3 h at 4 °C before harvest. Sampled protoplasts were lysed in 100 μl lysis buffer (Promega, E4550). After centrifugation, the supernatant was used for glucuronidase (GUS) and firefly luciferase (Luc) activity analysis. GUS activity was assayed with 1 mM 4-methylumberlliferyl-β-d-glucuronide in lysis buffer. The reaction was terminated with 0.2 M Na₂CO₃ after 30 min, the reaction product MU was measured using Fluorescence FLx800 microplate fluorescence reader (BIO-TEK Instruments). LUC activity was also determined by Fluorescence FLx800 microplate fluorescence reader. Ratios of GUS to LUC activities were used to define relative promoter activity. Three biological replicates, each with five technical replicates, were assayed for each construct. The cis-element analysis was conducted in PLACE (http://www.dna.affrc.go.jp/PLACE).