Domain specific interactions of S-RNase binding protein with stylar 120 kDA glycoprotein in Nicotiana [abstract]

Abstract

Through the process of evolution many flowering plants have developed a biochemical mechanism to prevent self-pollination and pollination by closely related plants. Gametophytic self-incompatibility (SI) is one such system that prevents inbreeding, a well-characterized disadvantage for organisms. A recently discovered protein S-RNase Binding Protein (SBP1) may be involved in SI in several species of flowering plants. SBP1 has been isolated in Nicotiana and has been shown to interact with the c-terminal of 120 kDa protein (120K), a key protein player in SI. It is unclear which domain or domains of SBP1 interact with 120K. In this experiment NaSBP1 has been cloned into pMAL-C2x an N-terminal fusion Maltose Binding Protein (MBP) expression vector. Using nucleotide primers it has been possible to amplify the desired NaSBP1 sequences for cloning into pMAL-C2x and expression in E. Coli. Transformants have been screened for expression using SDS-PAGE and western blotting. Clones that express the fusion proteins were sequenced to verify that no mutations in the DNA have occurred during PCR amplification. Binding experiments (pull-down assays) with domain specific MBP