The High Throughput Cloning, Expression And Preliminary Enzyme Activity Analysis Of Rice Protein Kinases

Rice (Oryza sativa.L) is an important food crop, which is an important genetic material in studying crop productivity, transgene, heterosis, and resistance. Rice is also a good model plant for the fundamental research. There are more than 1500 kinases in rice, as the biggest enzyme family that regulate post-translational modifications, playing important roles in all processes of growth and development. But the function-well-known kinases arefew.Generally cloning and expression of genes is the chiefly step to learn there functions. However the former cloning methods such as map-base cloning or transposon tagging which based on forward genetics can only cloned one or several genes at the same time. Now the completion of rice genome sequencing offers necessary condition for researching kinases' functions on genome levels, and also offers feasibility for large-scale cloning kinase genes.Based on reverse genetics, the primers were designed using the published rice kinase sequences. The encoding sequences of the protein kinases were obtained by PCR from the rice genome, and then cloned into pEGH vector using the yeast homologous recombination system. After PCR-based validation of the recombinating clones, the correct clones were induced to express in yeast by galactose. The expressed protein kinases were analysised autophosphorylation by [(?)-³²P]ATP. The following is the experiment conclusions:Base on rice protein kinase sequences without introns and transmembrane domains, 30 pairs primers were designed in this experiment;and 26 encoding sequences of these kinases were obtained through two rounds of PCR, enhancing the efficiency of transformation;Utilizing the yeast homologous recombination system, 23 recombinants were obtained;23 kinase proteins were induced to express in different levels;9 kinase proteins have the kinase activity by autophosphorylation assays.This work is helpful for antibody preparation, substrate screening, protein-protein interaction analysis, and provides an efficient system for high throughput cloning and expression of rice genes. This work is also provides a reference for large-scale researching other functional genes.