Expression Of XA21-LRR By Pichia Pastoris And Its Initial Analysis Of Glycosylation

Rice is a kind of very important crop, rice white blight disease is a serious bacterial disease which influence the development of rice seriously, it can even result in the lost of harvest. The studies to the mechanism of rice disease resistance gene Xa21, which confers resistance to Xanthomonas oryzae pv. oryzae have great significances. The studies can in favor of protecting rice from destroy, enhancing rice yield and solving the problem of world people's famine. We have known that XA21 mainly contains three domains, the Leucine Zipper, an extracelluar leucine-rich-repeat domain, which includes 23 different LRR domains, the transmembrance domain and a serine-threonine kinase-like domain. The LRR domains have been postulated to mediate cellular signaling process which maily recognize the ligands of AVR in rice defense responds. We have proved that the XA21 protein is glycosylated (The data is not published). Then we speculate that the LRR domains are glycosylated and act as signal antenna to recognize the inbreak of pathogenic.The research methods that we adopted are as follow. In order to express the protein of interest, the experiment is divided into two steps, First of all, we utilize secreting plasmid which come from Multi-Copy Pichia Express Kit to establish the recombinant vectors, after the vectors are established, we transfer the recombinant vector to Pichia pastoris , when we confirm that the vector has integrated into Pichia genome by a series of screening, such as auxotroph plate,YPD-G418 plate and PCR, we use methyl alcohol to induce the expression of recombinant Pichia strains, and analysis the target protein by SDS-Polyacrylamide Gel Electrophoresis and western blot detecting. Secondly, after the expression of the target protein, we deglycosylation of the secreting protein by PNGase F, and then after the reaction, the mixture were subjected to SDS-PAGE and Western blot analysis to identify the existence of sugar chain by the changes of mass. The binding sites of sugar chain on the target protein will be analysed by mass spectrum in the following steps. In general, we have expressed the protein of interest successfully. We use PNGase F to digest the sugar chains of target protein. We conclude that the protein of interest has sugar chains by western blot detecting. The next step of illustrating the situation of glycosylation on LRR domain, such as the binding sites of sugar chains on target protein, and the types of glycosyl is preparing. The experiment will play a certain role on the study of sugar chains, which may take part in the signal transduction pathway in rice defense responds.