| Constitutive promoter is one kind of promoters which is widely used in plant genetic engineering. This kind of promoter can drive target genes with a non-specific, sustained, stable expression pattern; but can not regulate the gene expression in a temporal or spatial manner. This may lead to a waste of plant nutrition, or even damage to the transgenic plants. Tissue-specific promoters or stress-inducible promoters can drive the expression of target genes to only the required tissues or at a specific time. So cloning and studying the tissue-specific or/and stress-inducible promoters will be important to provide new materials for plant genetic engineering. The promoter of TsNPT from Thellungiella halophila which had been cloned in our lab before was used in the present study. By analyzing the function of this promoter we identified a key region which is related to the shoot-specific and salt-stress inducible ability of this promoter. The upstream protein which binds to this DNA region was also identified. This result may provide important information for further study in the expression pattern of this promoter and the gene expression regulation mechanism in Thellungiella halophila.21 putative cis-elements were identified in the TsNPT promoter region by online analysis. They may be involved in multiple regulation pathways including stress induction or tissue-specific expression. To study the function of this promoter in detail, several 5'-deleted mutants were constructed, linked the GUS reporter gene and transformed into Arabidopsis. GUS staining of transgenic lines D1 in which the GUS reporter gene was driven by the full-length TsNPT promoter was performed and the results indicated that the GUS was obviously expressed. But the expression level differed a lot in different parts of the plants.The GUS activity in the leaves was much higher than that in the roots.While treated with salt stress, the GUS activity of the line D1 was induced, especially in the leaves. Although the GUS activity in the roots was also induced, the changing-fold of the roots was much lower than that of the leaves. This result was further confirmed by GUS enzymatic activity measurement.In order to further study the function of this promoter, GUS staining of different 5'-deleted mutants was carried out. According to the results, transgenic lines of D2 and D4 to D7 had obvious higher GUS activity in leaves than roots under normal condition. This was consistent with D1.But the GUS activity of D3 was obviously reduced compared to these constructs, especially in leaves. The GUS expression level of D8 was visibly lower than D7. The GUS activity was only found in the vascular tissue in D8. While treated with salt stress, the GUS activity in the lines of D2 to D7 was obviously induced, especially in leaves. This was consistent with D1,too. But this salt-response ability disappeared in D8.The GUS activity in D8 was not changed after salt stress. They still had only a basic expression in the vascular tissues. According to the results mentioned above, the 134bp region (-365 to-232) was related to the shoot-specific and salt-stress inducible ability of this promoter.The upstream binding protein of this promoter region was identified by yeast one-hybridization screening. The results indicated that the histone protein TsH3.2 can bind to this promoter region in the yeast. To further confirm this result, TsH3.2 was expressed in E.coli strain BL21 and purified. The purified TsH3.2 protein, the Thellungiella halophila nuclear protein and total protein was used to do the in vitro protein-DNA binding experiment. The result showed that the TsH3.2 protein can bind to the 134bp promoter region specifically. This result may provide important information for further study in the expression pattern of this promoter and the mechanism of gene expression regulation in T. halophila.
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