Cloning EeHKT1;4 Gene In Elytrigia Elongata And Its Transformation Into Tobacco

Elytrigia elongata is gramineous Elytrigia Desv. rhizomes thin plexus type herbaceous plant found in southeastern Europe and Asia Minor, and is a halophytic relative of wheat that is used to improve salt tolerance of wheat. Our previous studies showed SSR (simple sequence repeat) specific loci Xgwm044 was linked to salt tolerance of E. elongata, and shared high homology (93%) with high affinity K+transporter (TmHKT1;4-A2) from Triticum monococcum, named EeHKT1;4. HKT1;4 mediated Na+unloading from xylem sap in roots and sheaths to maintain low Na+concentration and high K+/Na+ratio in shoots. In this study, we cloned EeHKT1;4 from E. elongate. The expression levels of EeHKT1;4 in shoots and roots was quantificationally analyzed using real-time RT-PCR technique and the patterns change of Na+, K+ accumulation in shoots and roots by using an atomic absorption spectrophotometer was also investigated under various salt treatments; then EeHKTl;4 was transformed into the tobacco W38 using Agrobacterium tumefaciens -mediated leaf disc transformation methods, which laid the foundation for the role of EeHKT1;4 in salt tolerance of tobacco. The results were as follow:(1) The isolated cDNA was comprised of 1922 bp containing 28 bp 5’-and 227 bp 3’untranslated region (UTR) sequence and a 1722 bp open reading frame (ORF) with. The ORF encodes 573 deduced amino acid residues with estimated molecular mass of 62.92 kDa and a theoretical isoelectric point of 9.73, and had a region of a conservative sequence LFFTAVSAATVSSMSTV to code P-loop. It contained especially serine residues that are Na+ specific selective sites. It shared higher similarity with the known HKT1 (over 65%). Therefore, EeHKT1;4 belong to the family of HKT1.(2) The expression of EeHKT1;4 was mainly in roots, indicating it was not affected by external K+ concentrations (0-10 mM). Moreover, no significant differences in Na+, K+concentrations in roots or shoots was found between various concentrations of KC1.(3) The expression of EeHKT1;4 and Na+concentration was significantly higher in roots than in leaf sheaths with the increase of external NaCl (25-200 mM), accompanying with the reduction in Na+ concentrations of leaf blade relative to roots and leaf sheaths. Furthermore, E. elongata accumulated larger amounts of K+ in leaf blade than in roots and leaf sheaths under salt conditions.(4) It have successful constructed pBI121-35S-EeHKTl;4-Nos vector, and then is transformed into the tobacco W38 by Agrobacterium tumefaciens-mediated leaf disc, finally screening for To generation of the transgenic tobacco plants using 50 mg/L kanamycin and PCR.Therefore, these results showed EeHKT1;4 was a specific Na+transporters, which could be unloaded Na+from the xylem sap of leaf sheaths and roots, and further prevent leaves Na+accumulation to toxic levels., thus playing an important role in maintaining the K+, Na+homostasis in shoots..