| The C-terminal binding protein gene OsCtBP-A was found to be down-regulated during the interaction between rice (Oryza sativa L cv. Nipponbare) and Xanthomonas oryzae pv. oryzae (Xoo). It might play some important role in this interaction. Our research was aiming at elucidating the function of OsCtBP-A of rice by obtaining the overexpression and RNA interference transgenic plants of this gene.1. Full-length cloning and sequence analysis of OsCtBP-A. The information of 5′terminal sequence was obtained using 5′RACE and the full-length cDNA of OsCtBP-A was cloned. Bioinformatics was utilized to analyze the structure of this gene and the character of its promoter. OsCtBP-A was located in chromosome 3 with 6 exons and 5 introns. The 1541 nt mRNA of OsCtBP-A encoded a 424 aa protein with a 2-Hacid_dh_C domain in N terminal. Sequence alignment showed OsCtBP-A protein shares a 63% identity with AN of Arabidopsis and IAN of Ipomoea nil. Furthermore, some putative cis-elements in response to ABA, SA, cold and drought were identified within the promoter of OsCtBP-A gene using PlantCARE software: G-box (related to ABA signal induction), TCA-box (in response to SA), LTR (related to cold stress) and MBS (related to drought stress). These cis-elements might participate in the response to signal molecules and stress.2. Temporal and spatial expression patterns of OsCtBP-A and its response to signal molecules. OsCtBP-A was expressed in all tissues during seedling, tillering and tasseling stages. Analysis of the expression level of OsCtBP-A gene in response to different signal molecules (SA, MeJA, ETH, ABA) also showed that OsCtBP-A transcript was induced drastically by ABA (with a nearly 9 times expression at 8 h after treatment), followed by SA. Thus, it was hypothesized that the expression of OsCtBP-A depended on ABA signaling transduction pathway, or was under the cross control of ABA and SA.3. Overexpression and RNAi analysis of OsCtBP-A. The overexpression and RNAi constructs of OsCtBP-A and vector control were transformed into the rice calli through Agrobacterium-mediated transformation. Forty-five overexpression (T0 generation), 75 RNAi and 75 pTCK303 transgenic lines were successfully obtained. Plants resistant to hygromycin B were confirmed to be positive transgenic lines by PCR and GUS analysis.RT-Q-PCR was used to detect the overexpression lines, RNAi lines and pTCK303 lines. The results showed OsCtBP-A was overexpressed and silenced well. Scanning electron microscope was used to detect the stoma of confirmed lines P1 (vector control), RI5 (RNAi), OE4 (overexpression). The stoma of RNAi line was drastically smaller than overexpression line and vector control.Xoo was used to inoculate the confirmed transgenic lines. RNAi line was a little more sensitive to Xoo than the other two lines. OsCtBP-A was presumably not the principle gene involved in the interaction between rice and Xoo. It may play a mild role during the interaction through the regulation of signal transducting networks of ABA or SA. The leaf width of transgenic plants were not changed by OsCtBP-A like what AN did in Arabidopsis. The expression of OsCtBP-A has a positive correlation with the roots development. The development of roots was inhibited in the RNAi lines while enhanced in the overexpression lines. RNAi lines were more sensitive to drought stress than vector control and overexpression lines.In conclusion, we have cloned the full-length of OsCtBP-A gene using 5′RACE and obtained transgenic plants. We have found clues of this gene's function in stress response and the interaction between rice and Xoo. Further analysis on the stress response of transgenic plants will help us to elucidate the function of OsCtBP-A in-depth. |
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