| Constitutive promoters such as CaMV (cauliflower mosaic virus) 35S and Ubi (ubiquitin) have been used extremely as useful tools in many plant transgenic researches. Because of lacking temporal and spatial regulation, constitutive promoters have a number of potential drawbacks in genetically improved crops. Tissue-specific promoter, a class of the most useful promoters, can drive gene expression in particular space and time. They not only increase transgene expression in specific organs or developmental stages, but also can avoid unnecessary energy waste which is resulted from the gene expression in unexpected plant organs. Therefore, the isolation, identification and application of specific promoters have become a key aspect in transgenic plant researches. PNZIP is a novel mesophyll-specific gene isolated from short-day plant pharbitis nil that encodes a protein with a leucine zipper motif. In situ hybridization experiment indicated that it was expressed specifically in leaf photosynthetically active mesophyll cell but not in other nonphotosynthetic tissues. In order to find stronger leaf-specific promoters for transgene expression in plants, we have isolated PNZIP promoter. Detailed investigations, such as 5′deletion analysis, 3′deletion analysis and tissue-specific expression were then carried out in order to elucidate the regulation mechanism of PNZIP promoter. In addition, a novel transcription factor that can interact with PNZIP promoter was also isolated from tobacco by yeast one-hybrid system. The main results were as follows: 1. To characterize the regulatory mechanisms controlling transcription of PNZIP gene, we isolated its promoter region from Pharbitis nil by adaptor PCR. Sequence analysis showed that the PNZIP promoter was 1415bp in length and there was an overlap region between the promoter and the cDNA sequence of PNZIP gene, indicating the reliability of the promoter (GeneBank accession number AF373414). Primer extension analysis showed that the transcription start site was located 122 nucleotides upstream of the translation start site of PNZIP gene and 30 nucleotides downstream of the TATA box. In addition, the proximal sequences of transcription start site conformed to the consensus motif for eukaryotic transcription start site. 2. In transgenic tobaccos, fluorometric GUS assay and Histochemical analysis of GUS activity show that PNZIP promoter is a specific promoter in photosynthetic tissues, and its GUS activity in leaf is 9-fold than that of 35S promoter and 2-fold than that of Rbcss-3A promoter. Moreover, PNZIP promoter is under circadian control with the highest activity at 4:00 and the lowest activity at 16:00, as opposed to Rbcs-3A promoter. 3. Sequence analysis revealed that several putative cis-elements, such as G-box, box-II, AT-like box, circadian-box exist in it. In addition, we also found another two putative new elements, such as GAAATA element and GATACT element, were present in PNZIP promoter. Both 5′and internal deletion analysis revealed that G-box and GATACT element were necessary to confer the leaf-specific expression of PNZIP promoter, but Box-II, AT-1 like element only were positive elements. 4. Electrophoretic mobility shift experiments demonstrated that GAAATA element can interact specifically with protein in nuclei from tobacco leaf, suggesting that this sequence is probably a binding site for transcription factors. In addition, the first "G", the fifth "T"and the sixth "A"in GAAATA element were important nucleotides that can be bind by tobacco nuclear extract. Oligomerization of GAAATA element and fusion with the Q5 promoter greatly elevated activity of Q5 promoter in transient expression assays with tobacco leaves. Based on these data it is likely that the role of the GAAATA element is a cis-acting enhancer element in PNZIP promoter. 5. A novel transcription factor, which can bind the -379 ~-100 bp region of PNZIP promoter, was isolated by yeast one-hybrid system. The corresponding full length cDNA contains 1529...
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