INVESTIGATION INTO THE GENETIC BASIS OF CAPSAICIN PRODUCTION IN PEPPERS USING NEXT GENERATION RNA SEQUENCING AND SYNTHETIC BIOLOGY APPROACHES

Capsaicin, a molecule synthesized by plants in the Capsicum genus, is popular for its ability to produce a sensation of burning in any tissue it encounters. The synthesis of capsaicin molecules is achieved through the capsaicin biosynthesis pathway. In this dual study, our goal was to insert two crucial genes, pun1 and pAMT, into a strain of Saccharomyces cerevisiae to allow capsaicin synthesis and perform Illumina RNA sequencing on seven pepper species of increasing pungency to identify other key or novel genes needed or related to capsaicin synthesis. We implemented a golden gate cloning strategy to insert our genes of interest into bacteria to then be cloned into yeast. We believe that successful insertion into our yeast strain was achieved for one of the genes, pAMT, but the other, pun1, appears to not be inserted. We hypothesize that correct insertion and expression of pun1 would achieve capsaicin synthesis alongside expression of pAMT, as these two genes would complete the missing parts of the capsaicin pathway. We identified five possible new gene candidates with unknown functions grouped together with similar expression to known genes present in the capsaicin pathway. These novel genes were identified as follows: CA01g11020, CA12g21630, CA09g00520, CA03g28900, CA09g15570. We also identified five regions of interest that showed similar trends in expression patterns that could contain new promising genes that are not known to participate in the capsaicin biosynthesis pathway