Research Assistant University of South Carolina Aiken Aiken, South Carolina
Body of Abstract: Transposable elements are segments of DNA that can move from one position in the genome to another. We are studying the transposition mechanism of the mPing element because it is actively transposing in the rice genome and is being developed into a plant gene discovery and genome editing tool. Our overall goal is to identify ways to increase mPing transposition. The target site duplications (TSDs) are three base pair identical repeat sequences created on both sides of the mPing element when it inserts into the genome. Changing the TSDs from TAA or TTA has been shown to significantly decrease transposition. The consensus sequence adjacent to the mPing TSD is TTA:TAA, suggesting that TA-rich sequences facilitate insertion into the genome. We hypothesized that these adjacent bases may also facilitate Transposase binding and mPing excision, so we predicted that there would be higher transposition when the mPing TSDs are flanked by TTA:TAA. Yeast transposition assays showed that mPing flanked by TTA:TAA excises more than a mPing flanked by GGT:ACC. To confirm these results in plants, we used a GFP reporter where mPing excision results in green fluorescence. GFP reporter constructs for mPing with adjacent TTA:TAA or GGT:ACC sequences were transformed into an Arabidopsis line that contained the ORF1 and Transposase genes. The resulting plants were analyzed by PCR and fluorescence microscopy to determine the relative frequency of transposition. Preliminary results show that when mPing is flanked by TTA:TAA, there is a clear increase in transposition. This suggests that the increased flexibility of TA-rich sequences facilitates transposition complex formation.