Development of Chimeric Genes for Functional Analysis of Arabidopsis thaliana Ecotype Lz-0 SRKC and SCRC Genes

Some plants can increase their genetic variability through self-incompatibility (SI), whereby they use the products of two polymorphic genes found at the S locus, SRK and SCR, to recognize and initiate rejection of self–pollen (carrying the same S haplotype). Arabidopsis thaliana has lost its SI trait, and of its three distinct haplogroups, SA and SB possess pseudogenes for both the SRK and SCR genes. In this thesis, the functionality of haplogroup, SC (ecotype Lz-0) is assessed in this still self-fertile plant. The sequences of AtSCRC and AtSRKC, which possess no obvious null mutations, were tested for self-recognition capabilities. Desired portions of the two genes were amplified using specific PCR primers that included added restriction sites for easy cloning into the appropriate Ti plasmid expression vectors (pNBSWSCRa and pNBSWBa, respectively). The SCR8pr::AtSCRC::OCS terminator and the AtS1pr::eAtSRKC KpnI-NheI::AlSRKb chimeric genes were used to transform Agrobacterium, which were subsequently used to transfect A. thaliana ecotype C-24 plants. Cross-pollination assays were performed to test the functionality of the AtSRKC and AtSCRC transgenes. During these assays, no SI response was observed between the AtSCRC and eAtSRKC transformants. However, an SI response was observed between AtSCRC transformants and plants transformed with the chimeric gene of its functional ortholog, AlSRK36 (Honors Thesis, Martin Berger). This indicated that the AtSRKC gene is a psuedogene, while the AtSCRC gene remains functional