Identification And Functional Analysis Of Two Rice Genes (OsBTF3 And OsCtBP-A) Regulated By Xanthomonas Oryzae Pv. Oryzae

Elucidation of functions of key genes involved in the interaction between rice and Xanthomonas oryzae pv. oryzae (Xoo) is of great help to understand the molecular mechanism of bacterial blight. In the previous study, cDNA-AFLP was employed to analyze the gene expression patterns of mock- and Xoo-induced rice suspension cells. Ten of 31 sequenced fragments showed homology to genes with known or putative functions involved in metabolism, pathogen response and signaling. 21 others did not show any homology to sequences with known functions. Among these genes, the expression of OsCtBP-A was suppressed during the interaction, whereas the OsBTF3 was induced. We obtained the accordance results when using real-time reverse transcription PCR to analyze the expression of the two genes in Xoo-inoculated rice plants. In this study, we seek to identify the function aspects of the two genes.Total RNA was isolated from rice plants and cDNA was synthesized by reverse transcription. Specific primers were designed according to the sequences of OsCtBP-A gene and OsBTF3. Both OsCtBP-A and OsBTF3 were amplified by RT-PCR and cloned into pMD-18T Easy vector and sequenced. The results indicated that the cDNA sequences of two genes were 100% conformity with the sequences from Genbank. Bioinformatic analysis indicated that OsBTF3 was located in the chromosome 3 with 5 exsons and 4 introns. Its 528nt mRNA encodes a 175 aa protein, containing a NAC domain, which suggests that it may have the similar function as the BTF3 transcription factor. OsCtBP-A is located in the chromosome 3 with 6 exsons and 5 introns. The mature 1140nt-length mRNA encodes a 424 aa protein, containing a 2-Hacid_dh_C domain, which suggests that it is a member of CtBP-A protein family.Full length cDNA was cloned into prokaryotic expression vector pET21b. The recombinant construct of pET21-BTF3 for OsBTF3 was obtained and transformed into BL21 (DE3 ) pLysS strain of E.coli. The interest protein was expressed successfully when induced by IPTGSubcellular localization of OsBTF3 was shown by GFP fusion technique. Expression vector 35S-OsBTF3-gfp which containing OsBTF3-gfp fusion gene was constructed by inserting OsBTF3 into 35S-gfp. The constructs was transformed into onion epidermal cells by partical bombardment, laser scanning confocal microscope was used to detect subcellular localization of the fusion protein gene. The result showed OsBTF3 was present maily in the nuclei.The over-expression construct pCAMBIA1300S-OsBTF3 and RNAi construct pCAMBIA1300S-OsBTF3 RNAi were transformed into rice (cv. Nipponbare) callus via Agrobacterium-mediated gene transformation, and plantlets resistant to hygromycin B were regenerated.A bindery vector for RNAi analysis of OsCtBP-A was constructed by insertionof OsCtBP-A gene fragment into pTCK303. The RNAi construct was introduced into rice (cv. Nipponbare) callus via Agrobacterium-mediated gene transformation. Plantlets resistant to hygromycin B were confirmed to be T₀ generation transgenic plants by PCR and GUS assay.