Analysis Of Biological Function Of Gene OsGF14-c

OsGF14-c (Oryza sativa GF14c) is a novel rice gene, which was cloned by screening the rice inflorescence cDNA library constructed in our laboratory with a specific probe. The product that the gene encodes shares a high sequence similarity with 14-3-3 --one kind of regulatory kinase-dependent protein. The full cDNA sequence of OsGF14-c is 1154 bp, which encodes 256 amino acids. In order to study the biological function of this gene in the growth and development of rice, we constructed antisense expression vector p35SCAMBIA1301F and two RNAi expression vector US, UT. By virtue of Agrobacterium-mediated transformation, these three vectors were introduced into pre-cultured rice calluses, respectively. Hyg-resistent calluses were collected and cultivated for genetic analysis. Amino acid sequence analysis based on BLASTp shows that OsGF14-c has 71% identity with yeast (Saccharomyces cerevisiae) 14-3-3 homologous BMH2 protein. The eukaryotic expression vector pdYES2 was constructed by inserting OsGF14-c ORF (Open Reading Frame) into plasmid pYES2. By LiAC-mediated transformation, pdYES2 was introduced into BMH2-disrupted yeast host strain GG3000 and the recombinant strain PYGG shows similar phenotype with BMH2-functioning yeast strain GG3001. As there exists a typical G-Box – a cis-element specific to higher plant, we assume that OsGF14-c may play some role in the transcription activity and thus be located at the nucleus of the cell. The transient expression of the OsGF14-c-GFP fusing gene in the epidermis of the onion shows that OsGF14-c is nucleus-located. The prokaryotic expression vector pGF-c2 was constructed by fusing OsGF14-c ORF into plasmid pMALC-2 and was transformed into host strain E.coli XL1-blue, a 70kD OsGF14-c-MBP fusing protein was expressed under 2% IPTG. Using amylose affinity chromatography, the 70KD OsGF14-c-MBP fusing protein was purified from bacteria for preparing specific anti-OsGF14-c monoclonal antibody. All the work will facilitate further functional analysis of OsGF14-c.