Cloning And Characterization Of High K~+ Transporter Gene From Nitraria Sibirica Pall

The Nitrari.L, a typical halophyte, belongs to Zygophyllaceae, which mainly grows in the desert with strong drought tolerance, salt resistance and anti-sand ability. Thus researching the mechanism of salt tolerance and developing salt-tolerant genetic resources in Nitraria L can provide theoretical and experimental evidence for the genetic improvement of crops and forest trees.High-affinity potassium transport protein (High affactive K+transpoter, HKT) located on the plasma membrane, through controlling plant reabsorption of sodium ions and maintaining a stable balance of intracellular Na+/K+to improve plant salt tolerance. The plant transports Na+ up through the xylem parenchyma cells under the transpiration pulling action, HKT1 uninstalls excessive Na+from the xylemby to the phloem by the way of symplasmic, then transports back to the roots, to prevent the excessive accumulation of Na+ in aboveground tissues.In the study, a High-affinity K+transporter gene (referred to as NsHKTl) and its promoter sequence were cloned from the halophytic plant, N. sibirica using Reverse Transcription (RT)-Polymerase Chain Reaction (PCR). The cloned NsHKTl cDNA contains 1866 nucleotides, with a predicted ORF of 1662 nucleotides. The predicted ORF encodes a protein of 553 amino acids, which has high homology to S. lycopersicum HKT1 protein. Using the TAIL-PCR technology, the promoter sequence of the NsHKT1 was isolated, and bioinformatics analysis was performed which contains two dry dehydration response elements,one salt induced element, one low temperature response element, four auxin response elements and so on, indicating that the NsHKT1 gene expression is regulated by the environment and growth factors. Real-time quantitative results showed that the expression level of the NsHKT1 in stem was higher than those in roots and leaves of Nitraria. L. When subjected to low temperature, NaCl and drought stress, its expression level was significantly increased.In order to further study functions and heterologous expression features of the NsHKT1 gene, pBI101-NsHKT1, PORE-pNsHKT1::GUS eukaryotic expression vectors and pET-32a(+)-NsHKT1 rokaryotic expression vector were constructed, and transgenic Arabidopsis were obtained via agrobacterium-mediated genetic transformation. Phenotypic analysis of transgenic Arabidopsis showed that NsHKT1::GUS fusion protein was mainly expressed in leaf veins, consistent with the function of uninstalling excessive Na+from the xylemby to the phloem by the way of symplasmic. Arabidopsis heterologous expression profile results showed transgenic plants has obvious superiority in growth compared with wild-type exposed to salt stress.Phenotypic analysis E. coli overexpressing NsHKTl under abiotic stress showed that under salt, drought and cold stress, it grows better than non-transgenic E. coli. These results will provide theoretical and experimental basis for analyzing NsHKTl expression characteristics, function and mechanism, and improving salt tolerance of crops and trees by using the gene.