Substrates Screening Of Protein Kinases And Biochemical And Expressional Analysis Of Cystatin In Rice

Plants would trigger a series of signal transduction pathways and accordingly make the correspongding physiological and biochemical responses when plants accepting the external environment and internal stimulus during the growth and development. In the signal transduction pathway, the receptor kinase or the receptor-like kinases play a very improtant role. According to the importance and specificity of protein kinases, the screening of its substrates has been gained lots of attention. We have obtained the binding proteins of Pi-d2 by yeast two-hybrid system, that is possible candidate substrats of Pi-d2, receptor-like kinase resistance to rice blast. We have done the phosphorylation in vitro. At the same time, we study the relationship between the proteins and XA21. Next, according the candidate substrates, for example, Pib3 (OC-I, encodes cystein protease inhibitor) and its homologous protein OC-II, those biochemical and expressional analysis have done in this study. The paper results as follows:1. Through the yeast two-hybid system, the laboratory of Wenyuan Song has get 12 binding proteins of Pi-d2. We further validate the trans-phosphorylation of Pib1C, Pib3, Pib4, Pib8 by Pi-d2. The protein metioned above could be transphosphorylated by XA21 too, a recepor kinase resistance to rice blight. Maybe there exists the overlap phenomenon and have some shared components during the plalnts resistance process.2. Pib1 encodes a kind of E3 ligase, deciding the substrate-specific recognition in ubiquitin degradation system, including a U-box domain and 5 Arm domains. Especially, U-box domain plays an important role for abnormal protein ubiquitin degradation. In this study, Arm domain could be transphosphorylated by the kinases. This indicates Pib1 may initiate cell death and resistant functions through kinase phosphorylation. This conclusion still needs further study.3. Pib3 encodes a inhibitor of cysteine proteinases. The recombinant protein is an efficient inhibitor of papain and its inhibition rate is approximately 50%. Protease inhibitors may prevent proteases of pathogenic bacteria from the degradation of host organizations. Once the necessary nutrition is not satisfied, the growthing and developing of pathogens would be strongly arrested, which may be one of the immune mechanisms for plants to resist to pathogens.4. Pib4 belongs to Ser/Thr protease inbibitor which belongs to different families with Pib3. Same to Pib3, it participates the regulation to disease resistance of plant defense response. 5. Pib8 is a member MAPKKK family which perhaps involved in immune regulation in plants.6. There are 12 cystatin genes (OC-I to OC-XII) in rice (Oryzae sativa L.). In this study, we also studied the primary biological function of OC-II. The research shows that OC-II combined with receptor kinase XA21 in vitro. This should be needed to further validate.Multiple substrates of receptor kinase have identified in this article. They have combined with the kinase in vitro , which a good foundation for further study on its function.