Isolation Of SsNHX1 From Halophyte Suaeda Salsa And Gene Engineering For Arabidopsis Thaliana

Isolation ofSsNHX1 from halophyte Suaeda salsa and gene engineering for Arabidopsis thalianaThe maladies caused by salt stress arise from the disruption of cellular aqueous and ionic equilibrium. The ions, especially Na+ accumulates excessively in the cytoplasm, it can be toxic to the metabolism and affect the regulation mechanism of the plant cell. To cope with salt stress, plants have developed a variety of adaptation mechanisms. One is the accumulation of the solute osmo-protectants, the other is, the mechanisms of ion homeostasis including Na+ extrusion system, or sequester Na+ into the vacuolar and regulate the ratio of K+/ Na+, especially in halophyte. Na+/H+ antiporter plays a role in this process. AtNHXl (encoding a vacuolar Na+/H+antiporter) and SOS1 (encoding a plasma membrane Na+/H+antiporter) were isolated from glycophyte Arabidopsis thaliana, the over-expression of AtNHXl in A. thaliana increases the salt tolerance of transgenic plants significantly. The role of the SsNHXl in halophyte Suaeda salsa is ion homeostasis. Further research was done through the over-expression in A. thaliana, perhaps it could help us to elucidate the salt tolerance of halophyte. We isolated SsNHXl from halophyte S. salsa and transformed into Arabidopsis. Gene engineering base on reduce Na+ content in cytoplasm creates a new approach to plant salt tolerance. SsNHXl perhaps plays an important role in regulate Na+ homeostasis of halophyte S. salsa, so over-expression of SsNHXl in A. thaliana may make progress of the research. It can attribute to elucidate the ion homeostasis of plant cell.In this experiment, we isolated the SsNHXl gene (GenBbank accession number: AF370358). For the Na+/H+ antiporter gene had been isolated from A. thaliana, Oryza saliva et al.. There was conserved fragment in different Na+/H+antiporter, we designed the primers according to the conserved sequence, amplified the short fragment of SsNHX1. We obtained the fragment of 5' and 3' ends by using the RACE method. Two gene's specific primers were designed, and SsNHXl cDNA fragment was amplified with RT-RCR. After cloning and sequencing analysis, we knew that it had high homology with other vacuolar Na+/H+ antiporter. SsNHXlcDNA encodes a protein of about 61 KD and composed of 1665 bp. In order to know the copy of the gene in the S. salsa, southern was done, the results indicated that the protein was encoded with a gene family, there were at least three genes in the family. Northern results suggested that the SsNHXl was induced by salt stress, especially in the leaves, it was correlated with its predicted function.Then the SsNHXl ORF was integrated into the expression vector pROK2, the integrated vector was introduced into A. thaliana by in planta transformation method mediated by Agrobacterium tumefaciens GV3101. Transformants were screened with the media containing Kanamycin (30mg/l). Twenty-three transgenic lines were obtained in the T2 generation, PCR analysis indicated that all lines had been intergrated of SsNHX1. Southern blot suggested that it had integrated into the Arabidopsis genome. We selected two lines who had different expression levels in Northern blot to continue further physiology measures. The results indicated the Na+ content increased in transgenic plant leaves, the salt tolerance increase maybe correlated with the increase ability of Na+ compartment.