Genetic Transformation Of Mz₂NHX₁ Gene From Malus Zumi To Arabidopsis Thaliana

There are soil salinization all over the world.As the urban population growing,urban land is being gradually increased.The soil physical and chemical properties ware made badly by unreasonable planting and irrigation,which prevent the development agricultural.Salt stress is one of the most important abiotic stress in plants,it mainly showed in ion poison and osmotic stress.The main way of plants resisting salt stress is the excessive intracellular Na⁺efflux and segregation in vacuole.And the process is completed by the Na⁺/H⁺antiporters.Now,growing salt-tolerant plants on the salinizaty soil is the most effective measure to control soil salinization.With the progress of biotechnology,enhancing plant salt resistance has become one of the effective way to improving sailinity soil by the means of molecular biology and salt-tolerant genes,based on the further understangding of plant salt-tolerance mechanism.Malus zumi is originated from north mountain of Japan,it is the hybrids of Malus sieboldii and M.manshurica.It was introduced to China in 1976,and was propagation by tissue culture in Tianjin agricutrual university in 1981.It`s resistance is stronger and could grow normally under high salt content.Beyond that,it is an excellent dwarfing rootstock of apple and landscaping tree in saline-alkali soil.Therefor,it has important significance to explore the molecular of it`s salt and alkali tolerance.A full-length cDNA Na⁺/H⁺antiporter gene?Mz₂NHX₁?was isolated from Malus zumi according to the homologous sequence of NHX₁?GQ503257?from root in M.zumi.Sequence analysis indicated that the cDNA sequence was 1865 bp in length,including an open reading frame?ORF?of 1635 bp,which encoded a predicted polypeptide of 544 amino acids.The Mz₂HX₁ nucleotide sequence shared high identity with Md NHX₁?GU338395?,Rh NHX₁?KC188664?and Pe NHX₂?ACU01853?reported plant vacuolar Na⁺/H⁺antiporters.The identity ware 95%,93%and 91%,respectively.The homology of its amino acid sequence shared with AtNHX₁?AAF21755?,RhNHX₁?BAD93487.1?and AgNHX₁?BAB11940?ware79%,87%and 79%,respectively.Its means that Mz₂NHX₁ was localized to the vacuolar membrane.And Mz₂NHX₁ was a vacuolar Na⁺/H⁺antiporter gene.The purpose gene and dual plant expression vector pRI201-AN-GUS DNA ware linked by building a plant expression vector,and expression vector was successful.Arabidopsis thaliana was transformed using expression vector of Mz₂NHX₁ by Agrobacterium mediated.The resistance plants was screened by Kan.GUS histochemical staining method showed that the colour of lear vein and roots of the transgenic plants was the most deep,the secondly was young leaves,the dyeing of stem was not obvious.Extracting DNA from transgenic Arabidopsis thaliana,PCR amplifiy was done by P3 and P4 primers.One 1635bp band of obtained,which agreeing with the sequence of ORF of Mz₂NHX₁.This preliminary showed that Mz₂NHX₁ gene was transformed and expressed successful in Arabidopsis thaliana.Obtaining resistant plants is the transform Mz₂NHX₁ gene.