The Cloning And Analysis Of ZomNHX2 Gene From Zostera Marina And Apple Tissue Culture With A Preliminary Study Of Salt-resistance

Zostera marina belongs to Zostera L., is a marine higher angiosperm. Zostera marina lives in the seawater which contents salt as high as 600 tendency, so the saltresistance of Zostera marina is considered to be well. That confirms that it is a perfect material for the research of the salt-tolerance genes and the development of salttolerance mechanism. Na+/H+antiporters play an important role in plant salt-resistance.Because the Na+/H+antiporters can take excessive Na+which in cytoplasm out of cells or differentiate them into vacuolars, reducing salt ions especially the Na+ poisoning cells, to improve the salt-resistance of plants.The vacuolar Na+/H+ antiporter(Zom NHX2) from Zostera marina was cloned by RT-PCR sucessfully, and we got related information by bioinformatics analysis. Then Semi-quantitative RT-PCR technique was preformed to analysize the tissue specificity of the Zom NHX2 expression. In the future, we will build expression vector for genetic transformation of apple seedlings to verify the function of the Zom NHX2.Concrete results are as follows:1. To design specific primers based on sequence information searched from the genbank, then cloned the Zom NHX2 by RT-PCR. We learnt that the length of the Zom NHX2 is 1 626 bp, that the Zom NHX2 can encode 541 amino acid. Predicting the protein molecular weight is 59.5 k Da.2. Amino acid sequence alignment showed that the Zom NHX2 displays higher homology with Na+/H+ antiporters from other species and the highest homology with Iris lactea. According to homologous comparison, we found the Zom NHX2 contains with the typical conservative areas(LFFIVLLPPI) of Na+/H+ antiporters, which is the binding sites of amiloride. Secondary structure prediction showed that alpha helix is the main secondary structure of Zom NHX2. Hydrophobic analysis indicated that the N-terminal of Zom NHX2 is hydrophobic, that the C-terminal is hydrophilic and that the protein is hydrophobic. By TMpred prediction, it is has been shown that Zom NHX2 protein has 10 transmembrane domains. Phylogenetic tree indicated Zostera marina has close genetic relationship with Iris lactea.3. Using semi-quantitative RT-PCR technique to analyze the expression specificity of the Zom NHX2 in stems and leaves, we found that the Zom NHX2 expresses muchmore in the tissue of leaf than that in the stem tissue.4. Set up apple regeneration system. The root, stem, leaf and cotyledon explants could only be formed callus induced by the hormone. While, the germ regenerated shoots in high efficiency by the induction of the culture medium which is MS+KT(1 mg/L)+ NAA(0.2 mg/L). In order to obtain the seedlings, rooting experiment was carried out, and 1/2 MS+NAA(0.2 mg/L) is the best medium for rooting.5. The apple seedlings was treated with Na Cl for resistance research. Found that as the higher concentration of Na Cl, as the longer stress of salt-treatment, the salt-injury rate of apple seedlings is higher. In conclusion, the suggestion is that choose 4 g/L-8 g/L Na Cl to treat transgenic apple seedlings to study the salt-resistance of transgenic apple seedlings.