Transformation Of The Mir159-gamyb Pathway In Nicotiana benthamiana And Using Ruby As A Reporter Gene.

Abstract

The small microRNA159 (miR159) strongly silences the expression of genes encoding GAMYB transcription factors. This gene is a regulatory gene that controls the transcription of multiple other genes. When miR159 is inhibited, the expression of GAMYB is activated, inhibiting plant growth, but also activating plant pathogen defens mechanisms, including the expression of CP1. The CP1 gene is used as a marker that indicates when GAMYB is expressed. Therefore, this project engineered the binding site of miR159 in GAMYB which was positioned under the inducible XVE promoter. By doing so, the expression of GAMYB would only occur in the presence of estradiol. Moreover, to monitor the activation of this pathway, a CP1-RUBY construct was made using golden gate cloning that produced a red color, indicating when GAMYB would be activated. However, this project is still ongoing research, which means that no conclusion can be made from this study yet. Several procedures such as RNA sequencing, estradiol induction and CP1-RUBY transformation still need to be conducted.