| Salinity is one of the major abiotic stress factors that adversely affect the growthand yield of crop plant. Under salt tolerance, high concentration of sodium in soils aretoxic to most high plants, severely affect the growth and development of plants. Thestrategies for plants eliminating the Na+toxicity include decreasing Na+influx,compartmentalizing Na+in vacuoles and increasing Na+efflux. Compartmentalizationand efflux of Na+associated with vacuolar membrane and plasma membrane Na+/H+antiporters. The processes driven by the proton gradient established by the tonoplastH+-ATPase and V-type H+-PPase or plasma membrane H+-ATPase. As a result of thismechanism, Na+concentration has decreased, which can not only avert the toxiceffects of Na+in the cytoplasm but also maintain the normal intracellular K+/Na+ration.In addition, Na+stored in the vacuole can be used as an osmolyte to help tomaintain cell turgor and enhance the capacity of water uptake, finally osmotichomeostasis has achieved.Halogeton glomeratus is one of an annual dicotyledonous plants belong toChenopodiaceae. They mainly distributed in Gansu, Qinghai, Xinjiang and Tibet,mostly existed in grassland, saline-alkali land of desert and semi-desert areas.Halogeton glomeratus is one kind of halophytes, which leaves and stems are highlycarnified. Studying the Halogeton glomeratus regulation mechanisms of salt toleranceusing molecular methods, seeking a large number of genes and promoters which havepotential applications in drought resistance and salt tolerance breeding will have apractical sense in enriching salt-tolerant gene pool and cultivating salt-tolerant crops.1. According to the conservative region of NHX1gene in other species, theprimers were designed. Vacuolar Na+/H+antiporter gene of Halogeton glomeratus(HgNHX1) was isolated by RT-PCR and RACE methods．The full length of HgNHX1cDNA sequence was1979bp, contained a317bp3’-UTR and an ORF of1662bpwhich encoded a deduced protein about61kDa and composed of553amino acidresidues. Bioinformatics analysis showed that the deduced amino acid sequencecontained an amiloride binding site, which is a highly conservative sequence in otherNHX1of plants. The phylogenetic analysis revealed that the deduced proteinHgNHX1has a high degree of homology with other dicotyledonous plants.2. According to the conservative region of SOS1gene in other species theprimers were designed. Plasma membrane Na+/H+antiporter gene of Halogeton glomeratus (HgSOS1) was isolated by RT-PCR method．The full length of HgSOS1cDNA sequence was3498bp, contained an3498bp ORF which encoded a deducedprotein about129kDa and composed of1165amino acid residues. The phylogeneticanalysis revealed that the deduced protein HgNHX1and HgSOS1formed two distinctclades, verified the distinction of their functions.3. Under200mM NaCl stress, the relative expression level of HgNHX1andHgSOS1were elevated. HgSOS1is more sensitive to salt tolerance than HgNHX1,HgSOS1may response to tolerance more quickly and its relative expression level ishigher.4. To characterize the regulatory mechanisms controlling transcription ofHgNHX1gene, three nested specific primers coupled with arbitrary degenerate primerto isolate the5’ flanking region of HgNHX1gene by TAIL-PCR (thermal assymetricinterlaced-PCR). Sequence analysis revealed that a1514bp upstream of thetranslation start ATG start codon was amplified. The PlantCARE and PLACE wereused to analyse isolated sequence. The results showed that besides the TATA-box andCAAT-box, a number of potential cis-elements and transcription binding motifs whichare related to stress responses were found, such as salt, dehydration, cold andwounding responsive elements. Other potential cis-elements which are responsive tophytohormones, such as auxin, abscisic acid, gibberellin and ethylene, were alsofound in the sequence. Analysis of the sequence indicated that it has generalcharacteristics of typical promoter. |
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