Rice Integrin Immunohistochemical Localization Of The Protein And Its Encoding Gene Cloning And Expression

As cell adhesion molecules in animal, integrins belong to a kind of transmembrane protein superfamily. They are heterodimers which consist of two transmembrane glycoprotein subunits (αandβsubunits) and link by non-covalent bond. Integrins are important components of extracellular matrix (ECM)-plasma membrane-cytoskeleton continuum, and they mediate attachment between cells and cell signal transduction in both directions. The proteins which have the similar structure and functinon to integrins are called integrin-like proteins in plant. There is a homologous sequence between Os03g0199100 and AT14A, and the prediction of protein structure show that it is similar to one of the subunits of integrin in animals. In this study, we wanted to make clear that whether there was integrin-like protein in rice by biochemical, cell biological and molecular biological techniques. The main results were summarized as follows:1. Cell suspension cultures of Oryza saiva have been established preliminarily by improvement of nutrient medium and culture technique. We found that the concentration of 2,4-D and the pH of the liquid nutrient medium are the most important infactors for establishment of cell suspension cultures. The suspension cells cultured in medium with 2,4-D 2.0mg/L and pH5.3 grow well.2. Using a monoclonal antibody againstβ1 subunit of human integrin, immuno-fluorescent localization results, showed that the integrin-like protein localized in both cell membrane and nucleus in root cells,suspension cells and calli .3. The full-length sequence of Os03g0199100 was cloned from Oryza sativa by RT-PCR and TOPO-cloning technology. The Os03g0199100 gene was further fused with GFP coding sequences, and introduced into the wild type Oryza sativa by Agrobacterium-mediated method. Expresson of OsILP was analyzed by detection of GFP fluorescence. The results showed that OsILP targeted mainly to the plasma membrane at the cell level.According to the results above, there is an integrin-like protein mainly located in cell membrane in Oryza sativa.