| Promoter is a critical DNA element required for accurate transcription and regulation of transcription. In transgenic plants, promoter is one of the important factors affecting the efficiency of transgenic expression, so it is very important to choose the effective promoter. So far, many ubiquitin promoters from plants have been identified and used in transgenic plants because of their high efficiency, low levels of methylation, and the stability of genetic traits. The high-level expression system will be obtained by constructing the high efficient expression vector which were drived by this promoter. Thus, clone and utilization of the ubiquitin promoter will play an important role in the genetic engineering.A number of ubiquitin-extension protein gene sequences of different species were screened from Genbank, such as Arachis hypogaea, Nicotiana tabacum, Solanum lycopersicum, Capsicum annuum, Culex quinquefasciatus, Oryza sativa, etc.. According to the conserved homology region, nested primers were designed for anchored PCR to clone the upstream sequence of Chrysanthemum ubiquitin extension protein (UEP) gene. A 1472bps in length of the upstream sequence of the UEP gene was isolated by anchored PCR, which named DgUEP (Genbank NO. EU862325).Online bioinformatics software on the Neutral Network Promoter Prediction was used to analyze the core promoter element and transcriptional start site of the promoter sequence. The results indicated that there existed three core promoter elements located between nucleotides -1286 bp - -1236 bp,-318 bp--268 bp and -98 bp- -48 bp relative to the , translational initiation codon ATG (+1), and the possibility were 0.93, 0.93 and 0.95, respectively. The 1.47kb sequence of DgUEP promoter was scanned for cis-acting element using the web-based software program, PlantCARE and PLACE. The promoter appears to have several putative regulatory elements involved in pollen-specific expression and other elements include responsiveness to hormones, such as, the anther box like, TACPyAT-box, POLLEN1LELAT52, G-box, MBSII and so on.The plant expression vector which contain the DgUEP promoter and reporter gene,β-glucuronidase (GUS), was constructed and introduced into tobacco leaves and chrysanthemum petals for transient expression via Agrobacterium-mediated transformation. The results showed that the promoter can drive GUS gene's expression in tobacco leaves and chrysanthemum petals which demonstrated that this promoter have transcriptional activity. According to promoter DgUEP possible transcriptional start site and flower-specific expression of cis-acting element and other characteristics, 3' and 5' deletion mutation of DgUEP were acquired by PCR technology, and transient expression analysis of each mutantion was in progress in tobacco leaves and chrysanthemum petals. Histochemical and fluorometric analysis of GUS activity showed that the smallest fragment (-404 bp - +4 bp) of 3' end and the smallest fragment (-1457 bp - -1027 bp) of 5' end have transcriptional activity. The results indicated that the promoter may contain multiple transcriptional start sites. Fluorometric analysis of GUS activity showed that the main transcriptional start site of this promoter may be located between nucleotides -1457 bp - -1027 bp relative to the translation initiation codon ATG (+1). There may be some negative regulatory elements in -1027 bp - -604 bp, and some enhancer elments in -1457 bp - -1027 bp fragment. The activity of DgUEP promoter in the petals was significantly higher than the activity in the leaves. In conclusion, this study presumed that the promoter may be expressed mainly in the flowers. Furthermore, the expression vector contained the different promoter deletion of the DgUEP and GUS fusion gene were transformed into tobacco for stable expression via Agrobacterium-mediated, and some transgenic tobacco seedlings have been gained.
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