The endosperm is one of the two fertilization products inside the seed of most flowering plants. It surrounds and nourishes the embryo by providing nutrients. In most flowering plants such as Arabidopsis, endosperm development is made of a syncytial phase and a cellularized phase. After endosperm cellularization, the embryo growth rate increases while the endosperm growth rate decreases. Invertase inhibitor, InvINH1, is an enzyme inhibitor that suppresses growth. During the syncytial phase, embryo growth is slower, which correlates with higher InvINH1 expression in the micropylar endosperm. Our data indicated that the expression of InvINH1 is activated by transcription factor AGL dimers in the protoplast transient assay system. Since CArG cis-element is the conserved binding site for AGLs, the goal of this project is to mutagenize the four putative CArG sites in a 100 bp region of the InvINH1 promoter. Four pairs of primers were designed to amplify a vector containing the InvINH1 promoter while introducing the mutations. Sequencing data confirmed that all four CArG sites were mutagenized successfully. The mutagenized promoters will be tested in protoplast transient assay. If the CArG sites are required for the AGL dimer to bind InvINH1 promoter, the promoter activity is expected to decrease in the mutagenized InvINH1 promoters.
