| It has been demonstrated that protein kinases play important roles in almost all biological process, e.g. growth and development, regulation of expression, stress responses and signal transduction. These kinases are located on the node of various signal transduction process, forming a complex and orderliness metabolic nets together with the upstream and downstream interrelated proteins, regulating the plant growth and development and responses against environmental stress. Based on rice genomic sequences, bioinformatics analysis of homology sequences ofkinase domain suggests there are at least 1661 kinases in rice genome. Whereas, owing to the importance and particularity of kinases, cloning and expression kinases have becoming the main parts of functional genomics. Based on rice protein kinase sequences obtained from the database, kinases of functional representative and intron-free within gene were selected, 32 pairs primers were designed in this experiment; coding sequences of these kinases were obtained by PCR and cloned into yeast expression vector pEGH. GST-fusion proteins were expressed in Saccharomyces cerevisiae Y258, and their autophosphorylation activities were detected furthermore. The results are as follows:1.30 rice kinase coding sequences were obtained by PCR.2. 27 GST fusion proteins of kinase were expressed in S. cerevisiae with visible band after separated by SDS-PAGE and stained by Coomassie Blue. The expressed protein concentrates were about 0.5-5mg/L.3. Classification and functional annotation of kinases have been studied: according to the kinases Classification in PlantsP (http://plantsp.genomics.purdue.edu/), 27 kinases belong to 7 groups in the class 1 of Transmembrane Receptor Kinase and Related non-Transmembrane Kinases on PlantsP, namely Receptor like cytoplasmic kinaseⅦof group 1.2, CRPK1 Like Kinase of group 1.3, Crinkly 4_Like kinase of group 1.4, Plant Extemal Response Like Kinase of group 1.6, CRPK1 Like Kinase (Types 1 and ) and S-Domain Kinase (Type 2) of group 1.9, Legume Lectin Domain Kinase of group 1.11, Wall associate kinase of group 1.5, respectively.4. 21 kinases expressed in S. cerevisiae can autophosphorylate in vitro assay. They belong to 7 groups as above, respectively. Among 21 kinases, Os07g38230,Os07g03810,Os01g06280,Os03g21540,Os04g52860,Os03g12470,Os01g48000,Os01g65030 and Os03g61310 show strong autophosphorylation, which belong to Legume Lectin Domain Kinase,Receptor Like Cytoplasmic KinaseⅦ,CRPK1 Like Kinase,Wall associate kianse and S-Domain kinase.5. Juxtamemebrane domain is necessary to phosphorylation for almost plant receptor- like kinases.Kinases usually play various roles through the substrates phosphorylated, it is important to screen substrates of kinases for metabolic control nets and elucidate functional mechanism of kinase in cell. The rice blast resistance gene Pi-d2 encodes a Ser/Thr kinase different from other resistance gene reported in plants. Researching its anti-disease mechanism were important to theory and application aspects. Using the fusion proteins induced from rice cDNA and protein chip as a candidates, the kinase substrates were selected from the transphosphorylation assay in vitro with PI-D2 expressed in Y258. Whereas, one of the PI-D2 binding proteins with yeast two hybrid was phosphorylated by PI-D2 in vitro. The results are as follows:1. By solid phosphorylaion assay, 6 potential PI-D2 substrates were screened from cDNA expression library.2. The sequence ofα5 subunit of 20S proteasomes of rice was cloned, and its fusion protein expressed in E. coli.3. In vitro transphosphorylation assay,α5 subunit of 20S proteasomes was phosphorylated with kinase encoded by rice blast resistance gene Pi-d2.4. PIB1 is one of PI-D2 binding proteins from yeast two hybrid. In vitro transphosphorylation assay, PIB1 was phosphorylated by rice blast resistance kinase PI-D2, PIB1 encodes a U-box/armadillo repeat protein.5. Screening PI-D2 substrates through phosphorylation was carried out on rice protein chip designed by our laboratory, and many potential kinase substrates were obtained.The systems of high throughout cloning, expression and screening substrates of rice protein kinases were set up. In this study, these results provide critical bases to the study of anti-disease, anti-stress, development and regulation and kinase functional genomics. |
