Expression Of XA21 And PI-D2 Kinases In Yeast Expression System And Their Autophosphorylation Assay

Rice (Oryza sativa) is a very important crop, rice disease can reduce the yield office, it can even resμLt in the lost of harvest. Rice bacterial blight and Rice blast are the most important rice disease.The study to the mechanism of rice disease resistance gene has great significances, The study can benefit us of protecting rice from destroy, enhance rice yield and solve the problem of world people's famine. Xa21, which was cloned by Song et al in 1995, is a resistance gene of rice bacterial blight. Rice bacterial blight was caused by the bacterial pathogen, Xanthomonas oryzae pv. Oryzae, Xoo. We have known that XA21 mainly contains three domain, the Leucine Zipper, an extracelluar leucine-rich-repeat domain, which includes 23 LRR, the transmembrance domain and a serine-threonine kinase-like domain. The Pi-d2 is the resistance gene office blast. Rice blast, caused by the fungal pathogen, Magnaporthe grisea, is one of the most devastating rice diseases in the world. The dominant resistance gene, Pi-d2 (previously named Pi-d (t)2), in rice variety Digu conferring gene-for-gene resistance to a Chinese blast strain, ZB15, was previously mapped closely to the centromere of chromosome 6. Both are the receptor kinase-like proteins. Protein kinases are essential for the regμLation of cell growth and development in Rice. The characteristic of the receptor-like kinase XA21 has been researched systematically in the latest research in bacteria expression system. To acquire mass proteins of kinases XA21 and PI-D2, the S.cerevisiae yeast expression system is used to express, purify, analyse autophosphonylation activity. The experiment establishes the foundation for the research of the interaction of protein and protein, biochemical prosperities analysis and substrate screening.The method that we practiced in detail are as follow. First of all, in the S.cerevisiae yeast expression system, based on rice resistance gene Xa21 and Pi-d2 sequence, the primer sequences were designed by computer software.Xa21 and Pi-d2 kinase sequences were obtained by PCR.Recombine the DNA seggments and vector pEGH in the S.cerevisiae yeast strain.After identify and ensure its validity,Expression of target genes in host Yeast strain induced by galactose and analyse autophosphonylation activity, secondly, we utilize MμLti-copy Pichia express system to express my target proteins, after the vector established, we transfer the recombinant vector to Pichia pastoris, when confirm that the vector has integrated into Pichia genome by a series of screening, such as HIS- plate, YPD-G418, YPD-Zeocin and PCR, we use methyl alcohol to induce the expression of recombinant Pichia strains, and analysis by SDS-Polyacrylamide Gel Electrophoresis and western blot. In general, we have expressed the protein of interest successfμtLly except the concentration of target protein is not high.