Isolation, Characterization And Expression Pattern Of A Vacuolar Na~+/H~+ Antiporter Gene TrNHX1 From Trifolium Repens L.

Salt stress is a major abiotic stress that affects the growth,development,yields of crops and surely the environment.Salinity has a great impact on K⁺ uptake, cytoplasmic enzymes activity,photosynthesis and metabolism of plants.Excess salinity causes water-deficit resulting in osmotic stress and ion toxicity,which are the main reasons for the retardarce of plant growth and development in salina.Vacuolar compartmentation of Na⁺ through Na⁺/H⁺ antiporters into larger vacuoles is an essential mechanism for salinity tolerance of plants.Firstly,sequestration of Na⁺ into vacuole could lower cytosolic Na⁺ accumulation,which keeps redundant Na⁺ from the metabolism sites and alleviates the toxic effects on enzymes and membrane system. Secondly,plant could take advantage of Na⁺ into vacuole as an osmotic regulator to lower the water potential of cells and to resist the osmotic stress of salt.Moreover,the accumulation of Na⁺ in vacuole is one of the most effective mechanisms for the maintenance of high K⁺/Na⁺ ratio in cytoplasm.Trifolium repens L.(white clover)is a prostrate legume used for grazing,pasture hay and ground cover in horticultural situations.A highly desirable feature of white clover in pastures is its nitrogen fixing ability,which makes it adaptable to a wide range of soil and environmental conditions and combines well with many perennial grasses.However,few studies have been focused on the improvement of its salt tolerance and no related salt-tolerant genes have been cloned and identified from Trifolium repens,which limits its application in genetic reconstruction.Na⁺/H⁺ antiporter gene is recognized as one of the most valuable salt-tolerant genes in the world.Therefore,it is an important research project to clone the Na⁺/H⁺ antiporter gene from Trifolium repens and to conduct functional analysis,which will deepen the understanding of its salt-resistant mechanism and improve its salt tolerance by means of genetic engineering.This work should be very helpful to breed new cultivars and varieties for animal husbandry,turf industry and ecological construction in many regions.By the mutiply alignment of amino acid sequences and nucleotides acid sequences of several plants vacuole membrane Na⁺/H⁺ antiporters in GenBank,a pair of degenerate primers was designed against the conserved regions which was used for RT-PCR to amplify a 288bp cDNA segment from the Trifolium repens L..Based on the sequence of the cDNA segment,gene specific primer was designed for 3' RACE and a 1475bp cDNA segment was acquired.Based on the sequence of 3' end of the gene,primer was designed for 5' RACE and a 1030bp cDNA segment was acquired. The full-length cDNA was 2394bp gained by overlapping sequences,and the analysis of the sequence indicates that it contains an open reading frame,comprising 541 amino acid residues with a calculated molecular mass of 59.5 kDa.The sequence of ⁸⁵FFIYLLPPI⁹³is identified as the binding site of amiloride which inhibits the activity of Na⁺/H⁺ exchanger.Multiple alignments revealed a high degree of homology between deduced amino acid sequence of TrNHX1 and putative vacuolar Na⁺/H⁺ antiporter of other high plants.The highest identity was found to be 93%similarity with that of Medicago sativa.The result demonstrates that the gene cloned from Trifolium repens is a putative vacuole membrane Na⁺/H⁺ antiporter gene named TrNHX1,and its GenBank accession number is EU109427.Yeast complementary test showed that the expression of TrNHX1 could partly restore the growth ofΔnhx1 in the presence of 0.5M NaCl,0.1M Li Cl and 1.5M KC1 andΔena1-4Δnhx1 in the presence of 0.1 M NaCl and 20ug/ml hygromycin.Real-time Q-PCR was employed by using actin as a referance gene to quantify the expression intensity of TrNHX1 in different tissues under salt treatment.The expression level of TrNHX1 in roots,shoots and leaves of the untreated and salt-treated plants exhibited the following patterns:treatment with 150 mM NaCl caused 1.7-fold,2.2-fold and 4.3-fold increase of TrNHX1 expression in roots,shoots and leaves respectively, comparing with that of the controls.That is,the expression increment of TrNHX1 was highest in leaves while lowest in root.The Na⁺ contents in leaves,shoots and roots of the untreated and salt-treated plants were measured by atomic absorption spectrophotometer with the same way of salt treatment.It turned out that Na⁺ contents in roots,shoots and leaves with NaCl treatment caused the increment of 1.14-fold 1.75-fold,and 2.31-fold respectively,comparing with that of the controls.The trend that the increment of Na⁺ contents was highest in leaves and lowest in roots was just in line with the conclusion drawn from the real-time Q-PCR,which revealed the tissue-specific expression pattern of TrNHX1. Taken together,TrNHXl functions as a vacuolar Na⁺/H⁺ antiporter and plays an important role in salt tolerance and ion homeostasis in Trifolium repens L.