Discipline: Biological Sciences
Subcategory: Plant Research
Room: Exhibit Hall
Mary-Alaysha Woodford - Center for Biotechnology, Fort Valley State University, Fort Valley, GA
Co-Author(s): Noah G. Feinberg, Abhaya M. Dandekar, University of California, Davis, CA
Unlike the more widespread anthocyanins, plants in the order of Caryophyllales achieve pigmentation in their tissues by accumulating an alternative class of pigments called the betalains. The betalains are natural, tyrosine-derived, water-soluble pigments that give various fruits and vegetables their bright red color, which potentially can serve as a reporter to track gene expression or to visualize transgenic events. Because every cell contains the amino acid tyrosine, exogenous application of tyrosine to tissues may not be required. We hypothesized that betalain would be a more convenient reporter gene than the aforementioned reporters. Such as GUS, GFP and anthocyanin It is visible to the naked eye without any need for special equipment. It does not require processing samples and it allows for continuously monitoring of events throughout the life cycle of an organism. Moreover, it is applicable to large plants grown under normal field conditions. RUBY is a synthetic reporter gene that encodes the three biosynthetic enzymes that convert L-tyrosine into the betalains. The expression of RUBY is sufficient to reconstitute the pathway in non-Caryophyllales plants, resulting in the visible accumulation of betacyanins without the need for excitation or expensive reagents. However, due to RUBY’s long sequence and physical size, it is not suitable for compact plasmids or to be used as a fusion protein. It also has less utility in tissues that produce anthocyanins. In this project, we developed three RUBY-based reporter genes with the goal of working around some of these challenges. We developed a new version of RUBY, flexiRUBY, that replaces the self-cleaving 2A peptides with flexible glycine-serine linkers. This is expected to increase reaction efficiency by maintaining close proximity to the biosynthetic enzymes. Additionally, we utilized two versions of the L-DOPA Dioxygenase (DODA) enzyme from sugar beets (Beta vulgaris) and RUBY itself, renamed as BvMIDAS and rMIDAS. The MIDAS genes are significantly smaller in size and were expected to produce the betaxanthins solely, resulting in a golden pigmentation that could be distinguished in red tissues. References: He Y, & Zhao Y. Technological breakthroughs in generating transgene-free and genetically stable CRISPR-edited plants. aBIOTECH 1, 88–96 (2020). He Y, Zhang T, Sun H. et al. A reporter for noninvasively monitoring gene expression and plant transformation. Horticulture Research 7, 152 (2020). Chung H, Schwinn K, Ngo H et al. (2015). Characterization of betalain biosynthesis in Parakeelya flowers identifies the key biosynthetic gene DOD as belonging to an expanded LigB gene family that is conserved in betalain-producing species. Frontiers in plant science. 6. 499.
Funder Acknowledgement(s): This study was supported, in part, by a grant from UC-Davis HBCU Initiative-PABGAP. The author would like to thank for financial support from NSF S-STEM (DUE-1834046), the Department of Education MSEIP (P120A2000016).
Faculty Advisor: Sarwan Dhir, email@example.com
Role: For this research, I prepared different types of media and needed antibiotics. Prepared glyceral stocks, and ran the different plasmids used through PCR to amplify the DNA in each strain. Placed the grown embryos onto selective media containing hygromycin to kill off any untransformed materials. Did in-fusion cloning and then ran the products through PCR. I performed PCR clean-up. Transformed and scree agrobacterium of the EHA105.