Endosperm is a tissue produced inside the seed of flowering plants. It surrounds the embryo and provides nutrients. It also provides signals to regulate embryo growth. Endosperm development consists of the syncytial and cellularization phases. Interestingly, embryo growth is faster in the cellularization phase than the syncytial phase. An invertase inhibitor, InvINH1, is highly expressed during the syncytial phase. Since InvINH1 inhibits invertase, an enzyme that promotes growth, the expression pattern of InvINH1 agrees with the slower embryo growth rate during the syncytial phase. The goal of the project is to identify the cis-elements in InvINH1 promoter that are responsible for its specific expression in the syncytial endosperm. To achieve this goal, 5’ promoter deletion constructs for the 1100 bp InvINH1 promoter were generated. Two InvINH1 promoter fragments (500bp, and 700bp) were amplified by PCR, digested with restriction enzymes XbaI and BamHI, and cloned into vector pUC-GUS via ligation. Post-ligation, the plasmid was transformed into E.coli cells. The clones containing the expected construct were analyzed and validated by restriction digestion. After further validation by sequencing, the promoter deletion constructs will be used in protoplast transient expression system to identify the region within the promoter that is bound by transcription factor AGLs.
