Cloning Of Vacuolar Na～+/H～+ Antiporter Gene From Suaeda Corniculata (ScNHX1) And Transformation Studies Of ScNHX1 In Medicago Sativa

Soil salinity is a major and important type of land, but the problem of soil salinityhas been aggravated by the wrong agriculture practices such as irrigation. Soil salinityis a major constraint to food production owing to its limitation of crop yield. The bestway for save this problem is transgenic plant in all the methods of harness the soilsalinity. With the development of molecular biology and the study of plant salttolerance, salt tolerance genes were isolated and transformed by DNA recombinationtechnology with the aim of improving the plant salt tolerance and making good use ofsoil salinity by culturing the new plant of salt tolerance. Vacuolar Na⁺/H⁺ antiportergene is a regulation gene of osmotic stress, it encode Na⁺/H⁺ antiporter protein. Thevacuolar transportation protein not only keep low level of Na⁺ in cytosol, but alsomediate osmotic balance in development of cell and organ, and regulate vacuolar pHand ion homeostasis. In this paper, a novel vacuolar Na⁺/H⁺ antiporter gene (ScNHX1)from Suaeda corniculata that grows in Baicheng region of Jilin Province wascloned (GenBank accession number: DQ512716). The plant expression vector ofScNHX1 was constructed and transferred to Medicago sative by Agrobacteriumtumefaciens-mediated leaf discs transformation. The main results are as follow:1. A vacuolar Na⁺/H⁺ antiporter gene (ScNHX1) is isolated from Suaeda corniculataby RT-PCR and 3' RACE and 5' RACE stragety,The full-length sequence contains1665bp, the entire open reading frame (ORF) contains 554anino acids.2. The functional and special structure of the vacuolar Na⁺/H⁺ antiporter gene wasanalysized:(1) ScNHX1 encodes a vacuolar Na⁺/H⁺ antiporter protein with pâ… value 7.47, thepredicted molecular mass is 61.2KD.(2) ScNHX1 gene had the highest sequence similarity with the vacuolar-typeNa⁺/H⁺ antiporter gene, but had the lower sequence similarity with the plasmamembrane Na⁺/H⁺ antiporter gene. The phylogenetic analysis of varioustonoplast Na⁺/H⁺ antiporters using DNAMAN software indicates thatScNHX1 is a vacuolar-type Na⁺/H⁺ antiporter gene.(3) By the TMpred software, the deduced amino acid sequence of ScNHX1contains 12 highly conserved transmembrane (TM) segments at the Nterminal and a C-terminal hydrophilic region. The sequence of⁸⁵LFFIYLLPPI⁹⁴ is highly conserved is in TM3, this region is the binding site of amiloride binding domain. TM5 and TM6 do not seem to span thetonoplast, which are considered critical for antiporter transport activity.(4) The deduced amino acid sequence of ScNHX1 contained Asn, testify theScNHX1 is a glycosylation protein.(5) The deduced amino acid sequence of ScNHX1 contained Asp in 141.3. ScNHX1 gene was constructed into the plant expression vector pBI121 andtransffered to Medicago sative by Agrobacterium tumefaciens-mediated leaf discstransformation.