Isolation And Characterization Of A Salt Inducible GhNHX1 Promoter From Gossypium Hirsutum

Gene expression and regulation are mediated by promoters in most instances, directly upstream to the coding DNA sequence by recruiting transcription factor, regulator and a RNA polymerase in a spatially defined fashion. Constitutive promoters such as CaMV (cauliflower mosaic virus) 35S have been used extensively for high levels expression of transgenes. As lacking temporal and spatial regulation, constitutive promoters have a number of potential defects in genetically improved crops. So tissue-specific promoters and inducible promoters are very important tools in plant genetic engineering. By far, few salt-inducible promoters are reported.Vacuolar-type Na+/H+ antiporters play an important role in plants salt stress tolerance, which can deliver Na+ into the vacuoles against the electrochemical gradient by vacuolar H+-ATPase and H+-PPiase. The compartmentation of Na+ provides an effective mechanism to avert the deleterious effects of Na+ in cytosol and maintain an osmotic potential by using Na+ in the vacuoles as an osmoticum.In previous study, We have isolated a Na+/H+ antiporter gene (designated as GhNHX1) from cotton (Gossypium hirsutum). GhNHX1 was strongly induced by NaCl and its overexpression improved the salt-tolerance of transgenic tobacco plants. In order to search salt inducible promoters for transgene expression in plants, we have isolated GhNHX1 promoter. Detailed investigations, such as 5'-end deletion analysis, EMSA, tissue-specific expression were then carried out in order to elucidate the regulation mechanism of GhNHX1 promoter. The main results were as follows:1. To characterize the regulatory mechanisms controlling transcription of GhNHX1 gene, we isolated its promoter region from cotton (Gossypium hirsutum) by TAIL-PCR. Sequence analysis showed that the GhNHX1 promoter was 1610bp in length and there was an overlap region about 49bp between the promoter and the cDNA sequence of GhNHX1 gene, indicating the reliability of the promoter (GenBank accession EF650649). Analysis of the GhNHX1 promoter using using PlantCARE and PLACE showed that a number of potential cis-acting elements involved in environmental signaling were present (Fig.1). The putative TATA box (ATATAAT) (Brassica oleracea ) was found at position -25/-19 relative to the putative transcriptional star site (TSS). Some MYB binding sites (WAACCA) and MYC binding sites (CANNTG) thought to be involved in cold or drought stress were also found.2. To identify functional cis-elements of the GhNHX1 promoter, full length promoter and a series of 5'-deletions were ligated to GUS (β-glucronidase) in pBI121 plasmid. Each construt was introduced into tobacco cells by Agrobacteriun tumefaciens mediated transformation, and GUS activity were tested with or without the presence of NaCl. The results of GUS activity showed that GhNHX1 promoter could be induced by NaCl and the highest GUS activity was examined at the region -52/+49 which was significantly induced by approximately 3.72-fold.3. To test whether the -52/-25 region of GhNHX1 promoter could be induced by NaCl,it was fused to 35S△46 (46bp of CaMV35S 3'-end, minimal functional core promoter) promoter in pBI121 plasmid, and the GUS activity results showed that The -52/-25 region was induced by NaCl treatment to 2.55-fold.4. Electrophoretic mobility shift experiments demonstrated that the -52/-25 region of GhNHX1 promoter could interact specifically with protein in nuclei from tobacco cultured cells. It indicated the presence of cis-acting element involved in salt stress response among this region.5. We generated transgenic tobacco plants with the construct of GhNHX1 promoter (-183/+49):: GUS. Histochemical staining showed that there was no tissue-specific feature of GhNHX1 promoter. GUS was expressed ubiquitously in leaves, stem, root of transgenic tobaccos after NaCl treatment.