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A Na~+/H~+Antiporter Gene, DmNHX1, Confers Salt Tolerance When Expressed Transiently in Nicotiana Benthaminana and Stably in Arabidopsis Thaliana

Author: XuYuan
Tutor: XiaTao
School: East China Normal University
Course: Biochemistry and Molecular Biology
Keywords: Na~+/H~+antiporter Salt tolerance Transient expression Transgenic Arabidopsis
CLC: S572
Type: Master's thesis
Year: 2012
Downloads: 128
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Soil salinity is one of major factors affecting plant growth, which could lead to ions imbalance、osmotic stress and oxidative damage. Salt stress is mainly caused by excessive Na+. Plants have formed three main strategies to sustain the ion homeostasis and avoid sodium toxicity in cytosol. These strategies include Na+extrusion, compartmentation of Na+into the vacuole and secretion of Na+.Na extrusion and compartmentation of plants are operated by Na+/H+antiporters which are loacated on plasma and tonoplast membranes. They are confirmed to play the important role in Na’homeostasis, pH adjustment and cell volume. It is suggested that compartmentalization of sodium into vacuolar through the vacuolar Na+/H antiporter is an efficient mechanism to avert the deleterious of Na+in the cytoplasm and to reestablish the new osmotic pressure. Many studies proved that overexpressing plant vacuole Na+/H+antiporter genes could play an significant role in plant salt tolerance.In our previous study, the vacuolar Na+/H+antiporter of Dendranthema morifolium was was isolated by RT-PCR using degenerate oligonucleotide primers and RACE method. Yeast complementary test showed that DmNHX1plays an important role in salt tolerance. In this research, the DmNHX1was transformed into Nicotiana benthamiana and Arabidopsis thaliana to further investigate whether this gene could enhance plant salt tolerance and its tolerance ability.Leaf discs assays of leaf tissue transiently expressing DmNHX1, which was a new method to test the activity of NHX, were performed to evaluate the potential ability of this gene to enhance plant salt tolerance. N. benthamiana leaf tissue was infiltrated with Agrobacterium transformed with the empty binary vector pGRAB or a binary, pGRAB-DmNHX1which were designed for the study of transient DmNHX1over-expression. After the two-day treatment to allow the expression occurred, discs were excised and floated on different sodium chloride solutions for5days. Salt treatment induced the leaf discs without expressing DmNHX1the bleaching along the leaf disc edge, and the bleaching increased with higher salt concentration, especially in350mM and500mM. In contrast, leaf discs expressing DmNHX1showed much less bleaching after the exposure to salt. To illustrate this, chlorophylls were extracted from the leaf discs and quantified by measuring the absorption of extracts at665nm and649nm to detect the degree of chlorosis. Levels of chlorophyll a, chlorophyll b and their summed values were calculated. Chlorophyll a and b levels were both reduced, with the most marked reduce in chlorophyll a, after salt treatment.Arabidopsis transgenic lines were produced to demonstrate the fuction of DmNHX1. Wild-type and transgenic plants had similar phenotypes when germinated on plain medium or medium containing50mM sodium chloride. In the presence of75mM sodium chloride, seedling size and root length was more markedly reduced for wild-type seedlings than that of transgenic plants. This change was more apparent in the presence of100mM sodium chloride. In addition, root length of all branches of the transgenic and wide plants was measured used WinRHIZO sotware, the length of transgenic plants was2.98cm whereas the wide type was just1.85cm at the present of75mM NaCl. Besides to the increased root length, the transgenic plant lines also showed more root forks than the control plants.The growth status of adult wild-type and transgenic plants were also observed with different salt solutions. After20days treatment, plant phenotypes were observed and plant heights and fresh weights were measured. Growth of wild-type plants was impaired, with reduced plant heights and fresh weights after treatment with100mM sodium chloride, and become dead under200mM sodium chloride. Differently, although the growth of transgenic plants was also impaired after100mM sodium chloride treatment, the status was better than that of wild-type plants. Moreover, transgenic plants could survive in200mM sodium chloride treatment although its growth was severely impaired.The transcript pattern of DmNHXl was analyzed in transgenic Arabidopsis under salt treatment. The result showed that the DmNHXl transcript level of transgenic plants increased after salt stress compared with that of non-transgenical control. At the present of50mM NaCl solution, the DmNHX1transcript level of transgenic line increased1.53fold (Dm T3) and1.26fold (Dm T8) compared with that of wide type plants. With100mM NaCl stress, the transcript level of transgenic line increased2.6and2.4fold respectively. This means the transcript levels of DmNHX1gene is directly to salt tolerance.In the absence of salt, there were no significant differences in any of the assessed ions between non-transgenic and transgenic plants. However, after salt treatment the transgenic plants accumulated high levels of sodium but less potassium than that of the wild-type plants, without major changes in calcium and magnesium ion contents. During the process of NaCl stress, transgenic plant accumulated more Na+(from2.922mg/g DW to65.589mg/g DW) than that of wide type control plants (from3.193mg/g DW to31.041mg/g DW). This data suggested that much of Na+are sequestrated into vacuoles in transgenic plants for maintaining cell osmotic equilibrium and causing little interference with metabolism for sustained plant growth.Totally, our data showed that the transiently expression in Nicotiana benthaminana and stably expression in Arabidopsis thaliana of the DmNHX1gene improved their physiological performance and salt tolerance. It suggested that a newly cloned DmNHXl would devote an extra data to ion transporters database.

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