Isolation And Characterization Of Na~+/H~+ Antipoter Gene From Dendranthema Morifolium

Salt stress is one of abiotic stresses that cause serious limitation on plant growth. Salinity imposes two stresses on plant tissues:the water-deficit and ion-specific stress resulting from altered K~+/Na~+ ration and Na~+ and Cl~- ion concentrations that inimical to plants,which can be observed as imbalance of ion homeostasis,metabolic disorder and ultimately delay or damage in plant growth.The two main mechanisms for plant salt tolerance are to maintain osmotic balance and low cytosolic Na~+.Restriction of Na~+,Na~+ efflux and compartmentation of Na~+ into the vacuole are three main strategies to avoid the toxic effects of Na~+ in cytosol.As indirectly active processes, Na~+ efflux and compartmentation are operated by Na~+/H~+ antiporter.Na~+/H~+ antiporters are membrane proteins located in plasma and tonoplast which play the major role in Na~+ homeostasis,pH adjust and cell volume throughout the biological kingdom,from bacteria,alga to higher plant and animals.The plant vacuolar Na~+/H~+ antiporter catalyses the exchange of Na~+ for H~+ against Na~+ gradient and moves Na~+ into the vacuoles.The compartmentation of Na~+ also reduces the water potential,avoiding osmotic stress caused by salinity.Since the first vacuolar Na~+/H~+ antiporter gene was cloned from Arabidopsis thaliana,researches on the gene is increasingly in-depth and many results demonstrate that overexpression of vacuolar Na~+/H~+ antiporter gene enables plants to confer better salt tolerance.Thus,vacuolar Na~+/H~+ antiporter plays an important role in salt tolerance.Chrysanthemum[Dendranthema morifolium(Ramat.) Tzvel]is one of four Chinese famous flowers,some of which are officinal.However,chrysanthemums are restricted within narrow scope as its limited salt tolerance.Based on the function of Na~+/H~+ antiporter,it is highly expected that overexpression of the vacuolar Na~+/H~+ antiporter of Dendranthema morifolium might provide a useful tool for the molecular breeding of salt tolerant chrysanthemum.In this study,the vacuolar Na~+/H~+ antiporter of Dendranthema morifolium was isolated by RT-PCR using degenerate oligonucleotide primers and RACE method. The DmNHXl cDNA contains 1897 nucleotides,with a 5'-untranslated region of 18 nucleotides,a predicted ORF of 1653 nucleotides,and a 3'-untranslated region of 226 nucleotides.The predicted ORF encodes a protein of 550 amino acids with a calculated molecular mass of 61.1KDa,in which contains the highly conserved fragment LFFIYLLPPI among other Na~+/H~+ antiporters.Based on alignment and phylogenetic analysis,DmNHX1 is a putative vacuolar Na~+/H~+ antiporter gene and the deduced amino acid shares 73%and 76%identity toâ–³tNHX1 and OsNHX1, respectively.A hydropathy plot analysis showed that DmNHX1 contains 12 putative transmembrane segments,which is also the typical feature of other NHX1 antiporters.To test the function of DmNHX1 via yeast complementary study,DmNHX1 was transformed intoâ–³nhx1,a salt sensitive yeast mutant strain.It was found that overexpression of DmNHX1 inâ–³nhx1 suppressed its observed phenotypes and caused partial growth recovery in NaCl(400mM),KCl(1M),LiCl(100mM) and HYG(50ng/mL) media which indicated that DmNHX1 plays an important role in salt tolerance.To analysis the expression pattern of DmNHX1,real-time PCR and Na~+ ion content measure were preformed between the roots,stems,leaves and flowers from salt stressed chrysanthemum and from control.The result of Real Time PCR showed that the transcript levels of DmNHX1 after salt treatment were increased 2.04-fold in roots,1.13-fold in stems,3.53-fold in leaves and 1.17-fold in flowers,compared to control,respectively.Na~+ contents measured by atomic absorption spectrophotometer rose up from 0.76%to 1.30%in roots,1.00%to 1.40%in stems,1.10%to 3.20%in leaves,respectively,whereas remains 1.40%in flowers.These data showed that the transcript level of DmNHX1 is NaCl-induced pattern,increased expression of DmNHX1 causes more Na~+ compartmented into vacuoles and the Na~+ content increases as a sequence of this compartmentation activity.Different degrees of increased transcript levels and Na~+ content after salt stress revealed a significant tissue-specific expression pattern of DmNHX1.Specifically,the transcript level of DmNHX1 was only slightly increased and the Na~+ content was almost without change in flowers after salt treatment,which suggested that Na~+/H~+ antiporter may play a peculiar role in flowers and was required for further study.