Promoters insertion into cloning vector
The results obtained from the in silico analysis are presented in the Project Modeling section.
Arabidopsis thaliana germination assays
Promoters insertion into cloning vector pSEVA234
The germination rates are shown in Table 1. These values were calculated as the quotient of the germinated seeds and the total number of seeds per plate.
|MS||MS + DMSO (µL)||MS + Spectinabilin (µg/mL)|
In Figure 1, it is shown a plate layout with both germinated and non-germinated seeds. Here, it is possible to observe that germinated seeds are easily identified by its green dicotyledonous. Also, we can observe a thin root growing out of the seed involucre with a closer look.
Overall, both DMSO and spectinabilin are responsible for a slight decrease in the germination rate, although this effect is not sufficient to compromise the germination and growth viability of Arabidopsis thaliana.
The phenotype analysis was performed through the observation of all seeds in a microscope. In Figure 2, the differences identified in roots of seeds inoculated in MS medium supplemented with DMSO or spectinabilin are depicted and compared with the MS medium.
Most seed roots which germinated in MS medium exhibit a curly and thick appearance. However, in the medium supplemented with DMSO or spectinabilin two distinct root forms are observed: one similar to the ones described previously and another one characterized by a thin and long filament appearance. Since spectinabilin is diluted in DMSO, it is not possible to conclude if the modified phenotype is a response only to the presence of DMSO or if spectinabilin also contributes to this aspect.
Both promoter constructions were amplified by PCR. To confirm the success of the PCR, we run an agarose gel electrophoresis. As shown in Figure 3, a band of the expected size is visible for both inserts which confirms both inserts were amplified by PCR.