Clonging And In Vitro Expression Of Rice Protein Kinases

Rice (Oryza sativa) is an important food crop, it is also a good model for the fundamental research. The amount of protein kinases is more than a thousand in rice. Rice protein kinases play diverse roles in a variety of processes, including development and defense responses. Cloning and expression of rice protein kinase genes are the first-line work to learn the function of these genes, and it is important for learning the mechanism of resistant response and the regulation of resistant process.Based on rice protein kinases cDNA sequences published, the primer sequences were designed by computer software. The coded sequence of aim kinases were obtained by PCR (Polymerase Chain Reaction) from rice seed cDNA library. Recombine the DNA segments and vector pEGH in Yeast strain. Transfer the recombinant to E.coli strain, then identify and ensure its validity. Expression of target genes in host Yeast strain induced by galactose. Detecting the purified protein by SDS-PAGE. By the bioinfor-matics means, we analyzed the nucleotide sequences of kinases and conclude its function. In this experiment. The results were summarized as follows:1. The improved method was adopted to extract plasmid DNA and plasmids mixture was obtained.2. Coding sequences of five rice kinases were obtained through two round of PCR. Reactions conditions are similar to different kinase.3. These five protein kinases have a high level expression under the optimized condition.4. The result of BLASTX had shown that PK1 is putative protein kinase HvPKABA1 in rice. It has 87% identity to protein kinase 1 in Aegilops tauschii , 85% identity to HvPKABA1 in Hordeum vulgare subsp.5. Analyse by BLASTX, the result be received that PK2 is protein kinase CKâ…¡ alpha subunit in rice. It has 91% identity to CKâ…¡ alpha subunit in Lolium perenne, 91% identity to CKâ…¡ alpha subunit in Triticum aestivum and 90% identity to CKâ…¡ alpha subunit in maize.6. The result of BLASTX had shown that PK3 is putative protein kinase-AFC1 in rice. It has 77% identity to protein kinase AFC1/AME2 in Arabidopsis.7. The result of BLASTX had shown that PK4 is rice putative serine/threonine protein kinase. It has 90% identity to salt-induced MAP kinase 1 in maize, 82% identity to MAPK4 in Petroselinum crispum and 82% identity to MAPK4 in Arabidopsis.8. Analyse by BLASTX, the result be received that PK5 is a calcium-dependent protein...