Funcitonal Research Of Receptor Like Kinase OsRLK399in Rice (Oryza Sativa)

Plant receptor-like kinases (RLKs) are transmembrane proteins, and are located on cellmembrane. The RLKs contain an extracellular receptor domain, transmembran domain and acytoplasmic protein kinase domain. Plant RLKs can be categorized into several groups based onthe protein motif in the presumed extracellular domains, such as LRR-RLK, S-domain RLK,EGF-RLK, Lectin-like RLK and so on. The RLKs are membrane receptor, and are responsible forrecognizing and receiving external signal. The RLKs could amplify signals by phosphorylation ofcytoplasmic protein kinase domain. The RLKs are involved in plant development, hormoneperception and response, plant resistance against all kinds of stresses.This research cloning a RLKs gene of S-domain subfamily, which is called OsRLK399.Through the bioinformatics analysis, revealed that the OsRLK399contains a extracellularS-domain, transmembran domain and a cytoplasmic protein kinase domain. So the OsRLK399isthe typical membrane receptors, it is possible that OsRLK399is involved in response to variousstresses, such as resistance against insect.We constructed a p35S:YFP-OsRLK399expression vector, and transformed into Arabidopsisprotoplast. The result show that OsRLK399is located on the cell membrane, also in the cytoplasm.So we presume that the OsRLK399expresses on the other membrane systems, for example, theendoplasmic reticulum. We analysis the expression level of OsRLK399in different tissues by theReal-Time PCR, finding which expresses in all the tissues and with a highest level in developingseed.The expression of OsRLK399in mRNA level was examined in rice seedlings in response tovarious stresses and hormone treatments by Real-Time PCR, in order to study whether OsRLK399is involved in plant resistance against insect feeding or other stresses and hormone response. Theresult indicate that OsRLK399has nothing to do with piercing-sucking mouthparts insect feeding,whether it is involved in chewing mouthparts insect feeding need further research. But the relativeexpression level of OsRLK399increase sharply after200mmol/L NaCl treatment, which wasinduced by NaCl treatment. We presume OsRLK399is involved in responses to salt stress, but thedetail still need further validation. We constructed overexpression vector of extracellular domain and the full length CDS ofOsRLK399, also the RNAi vector. From the extracellular domain overexpression transgenic rice,we have identified two positive homozygous transgenic line of rice by Western Blot. We find thatthe branch number of transgenic rice increased compared with the wild type rice by statisticsanalysis. Because of the branch number increasing, the total weight of transgenic rice increased. Inaddition, the plant height of transgenic rice increased.We assayed the wild type rice and transgenic rice with Sitobion acenae, and calculated therate of aphid repellency. The statistical analysis showed that there was no significant differencesbetween wild type rice and transgenic rice. So the insect resistance in rice is a complex processes.Many research reveals that RLKs usually form polymers, so we study the functional model ofOsRLK399by Y2H (yeast two hybrid). Our data shows that OsRLK399could not formhomodimer, which may form heterodimer with other proteins.The OsRLK399protein and the kinase domain protein of OsRLK399were expressed inBL21bacterial strain by the constructed expression vector. We purified the OsRLK399protein byNi-NTA Agarose, and next step we could identify kinase activity and autophosphorylation abilityof OsRLK399by phosphorylation assay.Rice is the most important food crops in our country and whole world. This study analysisthe function of a plant receptor-like kinase (RLKs) gene OsRLK399in rice, and provides a certainreference for rice yield and plant resistance against stresses. We believe that the study of the RLKswill get more and more in the future.