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HIGH THROUGHPUT CLONING,EXPRESSION AND AUTOPHOSPHORYLATION ANALYSIS OF RICE KINASES

LI Li Yun1 SUN Jian1 WANG Hai Jiao1 LIU Qian2 LIU Li Juan1 TAO Yong3 LIU Guo Zhen1(1College of life sciences, Agricultural University of Hebei, Baoding 071000; 2Hebei Academy of Agriculture and Forestry Sciences, Shijiazhuang 050051; 3Institute of genetics and developmental biology, Chinese Academy of Sciences, Beijing 100101, China)  
Protein kinases are essential for the regulation of cell growth and development in plant. Based on rice genomic sequences obtained from the database, bioinformatics analysis of homology sequences suggested over 1500 kinases in rice. In this study, 32 PCR primers were synthesized based on conserved domains of known kinases, 30 intron-free kinase PCR products were obtained, among them, 27 kinases were expressed in Saccharomyces cerevisiae with visible band after separated by SDS-PAGE and stained by Coomassie Blue. According to annotation, 27 kinases belong to 7 groups in the first class on PlantsP (http://plantsp.genomics.purdue.edu/), Receptor like cytoplasmic kinase VII, CRPK1 Like Kinase, Crinkly 4_Like kinase, Plant External Response Like Kinase, S-Domain Kinase (Type 2), Legume Lectin Domain Kinase, Wall associate kinase, respectively. Furthermore, 17 kinases showed enzyme activity detected by autophosphorylation experiment. The results provide critical materials to the study of biochemical prosperities and substrate identification.
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