Cloning And Characterization Of A High Affinity K～+ Transporter Gene From Aeluropus Littoralis

To maintain a high cytosolic K⁺/Na⁺ ratio in plants is likely to be one of the key factorsto enhance salt tolerance. Aeluropus littoralis grows normally without any toxic symptomsand can maintain a high K⁺/Na⁺ ratio under a high salt environment. Therefore we hope toobtain a K⁺ transporter which can high affinity transport K⁺ under a high salt environment, tomaintain the K⁺ nutrition and the balance of the K⁺/Na⁺ ratio, and the function of high-affinityK⁺ transporter in improving the salinity tolerance of plants would be studied.In order to isolate the full length cDNA of high affinity K⁺ transporter gene from theAeluropus littoralis, RT-PCR and RLM-RACE were carried out, and a pair of degenerateprimers were designed according the HAK homologous gene region of other plants. AlHAKcDNA (2884 bp) included a 300 bp 5′UTR, a 253 bp 3′UTR, and a 2331 bp open readingframe encoding a 776 amino acid polypeptide which was 90%, 83%, 82% to sequences ofHAK from Phragmites australis, Oryza sativa, Hordeum vulgare, respectively.The open reading frame of AlHAK was inserted into the yeast expression vector pYES2.0to obtain YES-HAK, and then to introduced to the Saccharomyces cereviae mutant strainsWâ–³3 (MATa, ade2, ura3, trp1, trk1::LEU2, trk2::HIS3), which was deficient in high affinityK⁺ uptake. A complementary study was carried out to determine the function of AlHAK.When expressed in S. cerevisiae Wâ–³3, Wâ–³3-HAK could grow under micromolar K⁺concentrations with a K_m value of 8μM, the growth of Wâ–³3-HAK was inhibited in thepresence of Rb⁺ and Cs⁺ but less affected by pH＜4.0, the data suggested that AlHAKtransporter was a high affinity K⁺ transporter. Compared with Wâ–³3-YES, Wâ–³3-AlHAKshowed less sensitivity to 300mM Na⁺.The ORF of AlHAK was inserted into the plant expression vector pBI121, and theresultant plasmid pBI121-HAK was transferred into Agrobacterium tumefaciens by the liquidnitrogen freezing thaw method, then transferred into tobacco (Nictiana tabacum L. cv. 89) viaby Agrobacterium-mediated method. PCR, Southern blotting and RT-PCR analysis showedthat the AlHAK gene was integrated into tobacco genome and expressed. Detections ofphysiology suggusted that the K⁺ nutrition and the salt tolarance of transgenic tabacoos wereascend: the contents of K⁺ in transgenic tabaccos were 2 times of wild-type; the transgenic tobaccos could root on MS medium containing 200 mmol/L NaCl while the wild-typetobaccos could not. In the condition of NaCl stress, compared with the wild-type tobaccos, thetransgenic tobaccos held lower Na⁺ content and higher K⁺ content, and thus remained a highK⁺/Na⁺ ratios; furthermore, photosynthetic analysis showed that the photosynthetic rate (Pn),transpiration rate (E), substomatal CO₂ concentration (Ci), and stomatal conductance (Gs)were all higher than those of wild-type; in addition, the free proline (Pro) and soluble sugarcontents of the transformants were both higher than wild-type, which maintained a relativelyhigher level of osmotic pressure and relative water contents; transgenic tabaccos exhibitedlower relative conductivity and higher activity of superoxide dismutase (SOD). The datasuggested that the AlHAK gene from A. littoralis encoding high-affinity K⁺ transporter mightimprove the salinity tolerance of engineered plants, and this is the first report to improve thesalt tolerance of plant which by overexpression of high affinity K⁺ transporter gene.