| Promoter is an important DNA sequence that regulates genes transcription inplant. To express foreign genes specifically, the specific promoter has been widelyused in plant genes engineering. Generally, constitutive promoters has often been usedin transgenic engineering to over-expression of transgenes. However, use of suchpromoters has often led to the negative effect on plant, such as altered morphology,reduced size and so on. Identification and analysis of these promoters is useful tounderstand the mechanism of genes expression and utilize in application of transgenicplant. Recently, many pathogen-inducible plant promoters were found and identifiedby computer prediction and experiment, at the same time, regulation function ofpromoters was analyzed. However, only a few of inducible promoters were reportedin rice.In previous research, Through cDNA-AFLP analysis of the genes expressionprofiling of interaction between rice (Oryza sativa L cv. Nipponbare) andXanthomonas oryzae pv. oryzae (Xoo), a rice transcription factor gene OsBTF3wasfound to be up regulated. So we concluded that OsBTF3could be on of the importantregulating factors in rice-Xoo interaction. Besides, OsBTF3has the features ofresponse to pathogen and signal molecule by real-time quantitative PCR. OsBTF3express differently induced by different things, OsBTF3was strongly induced by Xoo andABA.To develop a novel inducible promoter, bioinformatic was applied to analyse ofits2065bp promoter region. There were some putative cis-elements in this promoterby bioinformatic analysis of Plantcare and PLACE program. Promoter containscommon cis-acting elements and some elements including W-box, G box and MYB inresponse to pathogens and abscisic acid. In order to further understand the functionsof promoter, promoter OsBTF3and a series of its5' deletions sequence wereamplified and cloned by PCR from the genomic DNA of cv.Nipponbare. The35Spromoter of LUC-PUC19was replaced by promoter OsBTF3and5' deletionssequence, thus vectors were transferred into Arabidopsis protoplasts by PEG. LUCactivities from different constructs were assayed in a transient expression system ofArabidopsis protoplasts treatment by OD600=1.2Xoo infected8h, which revealed apositive regulatory region (-1584to-1180). In addition, similar assays with theseconstructs were performed to identify which part of the promoter was responsible for0.6mmol/L ABA induction. The results revealed the region between-1219and-1037was required for its induction by ABA.Further analysis of the positive regulatory regions, we found threeWBOXATNPR1, WRKY710S, and SEBFWNSSTPR10A elements in the regionbetween-1584to-1180. Further analysis of the positive regulatory regions, we founda conserved DPBFCOREDCDC3element in the region between-1219and-1037.Each cis-element and was cloned into the reporter vector fused with LUC gene andtested for their activities. The results showed that SEBFWNSSTPR10A and DPBFCOREDCDC3had stronger LUS activities than the full-length promoter, whentreatment with Xoo and ABA, respectively, suggesting they are the main players inthe region. Furthermore, we made point mutation in each element and did the assaysagain. The element carrying the mutated base lost response completely.To test whether the response of the cis-element SEBFWNSSTPR10A wasspecific to Xoo, we used different bacterial species including Pseudomonas syringaepv. tomato (DC3000), Acidovorax avenae subsp. citrulli (Aac), and Xanthomonasoryzae pv. oryzicola (Xoc). The LUC activity induced by DC3000was lower than thatof Xoo, while Xoc induced a similar level of LUC activity. In contrast, it had noresponse to Aac treatment at all, suggesting the response is somewhat related tobacterial species.In summary, we have identified specific regions in the promoter region ofOsBTF3, which are responsive to pathogen infection and ABA treatment, respectively.Therefore, Identification and activity analysis of promoter OsBTF3will affordscientific foundation for the mechanism of genes expression and application oftransgenic plant. |
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