Fluoview fv1000d ix81

To further determine the transcriptional activity regulated by promoter sequence of each component, the promoter sequences of DNA-S (+ 45 ~ + 226), −M (+ 45 ~ + 281) and -N (+ 45 ~ + 276) were amplified by pair primers (Table 4) and constructed into the upstream of GFP in GV1300 vector (Fig. 4a). The recombinant plasmids, GV1300, GV1300-S-pro, GV1300-M-pro and GV1300-N-pro, were separately transformed into the Agrobacterium tumefaciens GV3101 competent cells by freeze-thaw method and positive clones were subsequently identified by PCR. Then, a rapid, transient expression method of fluorescent fusion proteins in tobacco plants was conducted according to the references [44 (link)]. At 72 hpt, total RNA was extracted and reverse transcribed into the first-strand cDNA as described above. To detect the effect of different promoters on gene transcription, the relative RNA amounts of GFP were measured by quantitative real-time PCR (qRT-PCR) using SYBR Premix Ex Taq (TaKaRa, China). Three biological replicates were assayed. The internal control, GAPDH, was used to normalize each sample and GFP transcription level was evaluated based on 2^-△△Ct method. Student’s t-test was used to evaluate the differences. Meanwhile, the intensity of green fluorescence was observed by a confocal microscopy (FluoView FV1000D IX81; Olympus, Tokyo, Japan) observation under 488 nm and 546 nm.