Studies On The Differential Expression Of Plasma Membrane Proteins Under Salt Stress And The Function Of LRR Recptor Protein Kinase In Rice Root Tips

A highly purified plasma membrane fraction from rice Xudao 3 (oryza sativa) root tips was isolated by two phase partition system. Then we studied the changes of plasma membrane proteome under salt stress with IEF/SDS-PAGE two dimensional gel electrophoresis and separated the multiple span membrane strongly hydrophobic proteins with SDS-PAGE. The stress response proteins in plasma membrane were identified by MALDI-TOF/TOF and LC-MS/MS. Furthermore, the identified proteins were confirmed on mRNA level by real-time PCR analysis under salt stress. One of the identified proteins was a LRR Receptor Protein kinase. The functions of this protein were analyzed by Prokaryotic Expression of the N-terminal fragment, production of Polyclonal Antibody, western blot,immunohistochemistry, immunoelectron microscopy and immunoprecipitation in rice root tips under abiotic stress (such as salt, drought, cold and ABA treatment). The main contents of this dissertation are as followings:1. Research of plasma membrane comparative proteome and relative transcriptome in rice root tips under salt stress.The root growth of rice seedling was significantly inhibited by the 150mM NaCl stress for 48h. With or without 150 mM NaCl exposure for 48 h, a highly purified (90%) plasma membrane fraction from root tip cells of rice were isloated by two phase partition system (Dextran-PEG). Solubilized plasma membrane fractions were then analyzed by IEF/SDS-PAGE. Comparative analysis between three independent biological replicates revealed that the expressions of 34 proteins were changed after salt treatment, of which 16 were up-regulated and 18 were down-regulated, respectively. Mass spectrometry analysis indicated that most of them were membrane associated proteins which were involved in important physiological processes, (1) Membrane stabilization. It includes that remorin which possibly participate in the formation of membrane skeleton and cytoskeleton, Myosin heavy chain protein which is link protein in cell wall skeleton and promotes the synthesis of membrane callose by signal reception from cell wall, Solubleα-NSF attachment protein which possibly participate in transmembrane transport. (2) Ion homeostasis. It includes that E-subunit of V-Atpase and putative YLP which participate in the establishment of electrochemical gradient and iron transportation across the vacuolar membrane, HIR (putative hypersensitive-induced response protein) which participates in the control of K+channel,14-3-3 protein which regulates plasma membrane H+-ATPase activity and controls iron channel (e.g. K+efflux). (3) Signal transduction. It includes that CaM (Calmodulin) and DREPP which participates in Ca2+signal transduction, OsRPK1 which possibly participates in the signal transduction of environmental stress. High-salt solutions (0.6 MKC1) and gentle detergents (15 mMCHAPS) were used to wash plasma membrane proteome in order to reduce the complexity of the fraction. According to the results of the SDS-PAGE and ESI-MS/MS, many mostly hydrophobic transmembrane proteins were identified. For example, aquaporin PIP2.2 regulates water status in cell; annexin participates in transmembrane transport and maintaining the stability of membrane structure; plasma membrane ATPase participates in the establishment of electrochemical gradient across membrane; RabllC participates in vesicle transportation, etc. The research on mRNA levels for the proteins identified by the two-dimensional gel electrophoresis and SDS-PAGE was carried out by Real-time quantitative PCR. It was found that the changes of 5 proteins (putative remorin 1 protein, putative receptor protein kinase, hypersensitive-induced response protein,14-3-3 like protein, and cytochrome b5) were consistent between on the level of transcription and translation while 3 proteins (calmodulin, putative alpha-soluble NSF attachment protein, and putative YLP) were not as that. These results suggested that the changes on transcriptional level did not completely reflect the changes on translational level, which probably caused by the modification and stabilization of mRNA and protein.2.Function Analysis, Prokaryotic Expression and Polyclonal Antibody Preparation of the Extracellular Domain of Rice LRR Receptor-like Protein Kinase OsRPKl2D-PAGE study indicated a new LRR receptor-like protein kinase OsRPKl whose expression could be induced by salt stress in root tips of rice. Sequence analysis by the SMART software showed that OsRPK1 consist of three domains:signal peptide and six leucine-zipper extracellular domain, transmembrane domain, and cytoplasmic kinase domain. The Amino Acid sequence homology of OsRPK1 is 25.31%、22.7%、21.3%、23.07%、22.68%、20.47%and 25.05%with that of OsRLK1、OsLRK1、OsBRI1、OsXa21、AtRPKl、ZmRLK and DcPSKR. Phylogenetic tree of these receptor-like protein kinases shows that OsRPKl and ZmRLK have a more close relationship.In order to investigate the function of OsRPK1, we analyzed the antigenicity of OsRPKl by DNAStar Protean. The 154-350 Aa extracellular fragment of OsRPKl obtained by RT-PCR that has high antigenicity and specificity was subcloned into pET29a. The recombinant plasmid pET29a-OsRPK1 was transformed into E. coli BL21 (DE3) for over-expression in host strain. The expression level was about 30% of the total cellular proteins. After separated by SDS-PAGE, the target band excised from the gel was used as an antigen to raise the antibody in New Zealand rabbits. After separation, purification and Western blot of antiserum, we successfully prepared the anti-OsRPK1 polyclonal antibody with the titers of 1:20000. The results of western blot analysis showed that the antibody could specifically recognize the expressed fragment in E. coli and OsRPKl protein in root tips of rice. OsRPKl was proved to locate on the plasma membrane by Immunoelectron microscopy study. The expression of rice LRR-receptor protein kinase OsRPK1 were analyzed by using the obtained antibody under salt, drought, cold and ABA stress. The results showed that there was a significant increase in OsRPK1 expression after 12h of salt treatment as compared to the control, and the expression of OsRPK1 still remained a relatively high level until 48h after salt treatment, this result was confirmed by immunohistochemical analysis of the expression of the OsRPK1 in the root tips of rice seedlings under salt stress. Under drought stress, OsRPK1 expression kept a significant increase at 3h and 12h, then it returned to the control level at 48h. OsRPK1 was not sensitive to cold, which was only a slight increase after 48h of cold stress. Finally, ABA treatment induced significant increase of OsRPK1 expression. The research shows that: OsRPK1 is induced by osmotic stress, since drought and salt treatment cause osmotic stress in plants more directly than cold treatment; and OsRPK1 is involved in ABA signaling, because all three stresses are related to ABA production, and extracellular ABA induced OsRPK1 expression rapidly.