Cloning And Functional Studies Of Two Ros Regulation Related Rpk Genes In Rice

Receptor-like kinases (receptor-like kinases, RLKs) have been found in almost all theplants and paly crucial and distinguishable roles. RLKs have been studied and annotated to bemostly located on cell membrane. They are constructed by three parts: an extracelluLarreceptor domain, a single transmembrane domain, and a cytoplasmic protein kinase domain.RLKs are membrane receptor proteins and can be classified into different subfamiliesaccording to the differences in their extracelluLar domains. External signal are identified andreceived by the extracelluLar receptor domain, and then transmitted to the intracelluLardomain by the transmembrane domain. Finally, autophosphorylation and autologousdephosphorylated of the intracellular domain lead to signaling cascade. RLKs not onlyparticipate in the reguLation of normal growth and development of plants but also in thesignal transduction for biotic and abiotic stress responses.In the present study, we cloned two rice receptor-like kinases, OsRPK1and OsRPK2,which were identified from a rice OsNox2knockout mutant osnox2. Based on ourbioinformatic analysis, OsRPK2may be located on cell membrane, which is a typicalleucine-rich receptor-like kinases (LRR-RLKs), and has three alterative splicings, namedOsRPK2-T1, OsRPK2-T2and OsRPK2-T3, respectively. OsRPK1may be located oncytoplasm, which can not be attributed to already annotated RLKs subfamily and may be anew class of plant receptor-like kinase. It has two alterative splicings, named OsRPK1-T1andOsRPK1-T2.(1) Verification of OsRPK1and OsRPK2localization: The CDS of each alterativesplicing sequence were connected to P35S:: eGFP vector, then transferred the vestor into riceprotoplasts for transient expression. The results from confocal imaging showed thatOsRPK1-T1and OsRPK1-T2are localized in cytoplasm whereas, OsRPK2-T1andOsRPK2-T2are localiezed in cell membrane whilke OsRPK2-T3is localized in cytoplasm.(2) Expression patterns of OsRPK1and OsRPK2genes in different tissues and underdifferent treatments: Results of RT-PCR expression profile showed that OsRPK1andOsRPK2had varied degrees of expression in all tissues of rice throughout growing stages.Expression profiles of OsRPK1and OsRPK2under different environment factors were alsoinvestigated using both semi-RT-PCR and qRT-PCR methods. Firstly, hormone treatmentsindicated that the expression of OsRPKs in seedling root was induced by0.5mM SA,50M GA and10M MeJA, while expression of OsRPKs in stem was induced by0.5mM SA and5M MV and suppressed by50M ABA. Secondly, the expression of both OsRPK1andOsRPK2were stimulated in rice stems under38°C and4°C and by NaCl and CaCl2treatments. Thirdly, drought treatment with20%PEG markedly suppressed the expression ofthe two OsRPKs in root.(3) Expression of OsRPK1and OsRPK2in E. coli: Five alterative splicing proteins wereexpressed in BL21bacterial strain by the constructed expression vector. And then we purifiedthe two OsRPKs by Ni-NTA Agarose. This experiment can enable us to identify the kinaseactivity and autophosphorylation ability of the two OsRPKs in the next.(4)Acquirement of transgenic plants: We also constructed transgenic vectors ofover-expression, antisense RNA and RNAi of the two OsRPKs alterative splicings. Then,transgenic plants were obtained by method of agrobacterium infection. Phenotypeobservations indicated transgenic plants with over-expressed OsRPK1-T2were larger than thewild-type rice. Results of RT-PCR and qRT-PCR showed that expression of several genes inNOX family (i.e. OsNox2, OsNox3and OsNox7) were upregulated in anOsRPK1-T2-overexpressed transgenic plant, while the expression of other genes in NOXfamily (OsNox1, OsNox5, OsNox6, OsNox8and OsNox9) were downregulated in that plant.Transcripts of Rac family genes, in particular OsRac2and OsRac6, were downregulated inthe OsRPK1-T2-overexpressed transgenic plants. These results imply a complex regulatoryrelationship between OsRPK1and the NOX and Rac proteins in rice.As a whole, a transgenic system for over-expression and non-expression of the twogenes OsRPK1and OsRPK2has been established and the resulted transgenic plants will beacquired for further studies. In the next, based on our current results, the roles of the twoRLKs in ROS signaling and metabolism, the machanisms in cellular signaling transduction,as well as the functions in plant stress tolerance, especially to abiotic stresses such as drought,salinity and extreme temperature, will be well elucidated by employing a series of methodsand/or approachies including RT-PCR analysis, protein interaction examination, geneprokaryotic expression, transcriptome sequencing, bioinformatics methode and physiologicaldeterminations. Then, several key proteins that might directly interat with the two RLKs willbe identified and their functional ways in ROS signaling networks will be clarified. Based onthe studies of this research project, many useful and important evidence will be obtained forclarifying the biological roles of plant RLKs and their functional mechanisms in plant stresstolerance.