Cloning And Characterization Of Wall-Associated Kinase Gene Form Oryza Sativa And Pokaryotic Expression Of Its Kinase Domain

Wall-associated kinases （WAKs） are a type of receptor-like kinases which are known to directly bind to cell walls, which have structure domains of epidermal growth factor, transmembrane domain, intracellular serine/threonine kinase and two insertion position conservative introns in its genomic. The different members of the WAK gene family show distinct spatial and temporal expression patterns, and their expressions are induced by biotic and abiotic stresses. According to previous studies, WAK family members play important roles in signal transduction, pathogen resistance, heavy metals tolerance, response to mineral nutrition, cell expansion, and plant growth and development.Researchers had isolated 125 OsWAK gene family members from Oryza Sativa japonica cv Nipponbare, and previous results showed that OsWAK1 （Oryza Sativa Wall Associated Kinases 1） played an important role in plant defense response. However, little is kown about the the functions and mechanisms of WAK in invloving in plant growth and development and in defense response. Weather the OsWAK has the same functions and mechanisms in rice is also required to investigate. So, this study provided the basis to the WAK gene’s cloning and prokaryotic expression of the kinase domain. These results may lead to a better understanding of the physiological mechanisms of OsWAK. In this experiment, the main results were summarized as follows:1、The coding region of cell wall-associated protein kinase （OsWAK） gene from rice was isolated by RT-PCR using specific primers of rice wall-associated kinase sequence published, and its full-length is 2136 bp （NM₀01052189.1）. By the bioinfor-matics means, we analyzed the nucleotide sequences and putative protein sequences of WAK. The results showed that OsWAK included two introns and three exons. A multiple sequence alignment was 50% similarity with OsWAK and other proteins, including SbWAK from Sorghum bicolor, BdWAK from Brachypodium distachyon and TaWAK from Triticum aestivum. The putative WAK protein has structure domains of two epidermal growth factor （220～268 aa; 272～310 aa）, a transmembrane domain （324～343 aa）, intracellular serine/threonine kinase （393～662 aa） using SMART bioinformatics analysis softwares.2、Semi-quantitive RT-PCR analysis showed that OsWAK expressed in both roots and shoots. Expression of OsWAK can be induced by SA, MeJA, Cu2+, Al3+ and Na+, however, be inhibited by SNP.3、The kinase domain fragment of OsWAK was overexpressed in Escherichia coli as a SUMO fusion protein using the plasmid vector pSUMO. The recombinant plasmid named pSUMO-WAKD after identification and transferred to E.coli The results shows the expression of the 60-kDa fusion protein in E.coli cells transformed with pSUMO-WAKD after 0.2 mMol/L IPTG induction at 11℃. We obtained 0.6 mg/mL fusion protein by Ni-NTA resin chromatography, and WAK was obtained from SUMO-WAKD fusion protein by cleavage with SUMO protease, this provided experimental basis for measuring kinase activity.