| HKT (high-affinity K+ transporter)-like proteins function as a discharge of sodium and potassium absorption function and plays an important role in salt tolerance in plants. In this study, wheat TaHKT8 was studied at different levels, including gene cloning, bioinformatics analysis and eukaryotic expression. The main results are as follows:1. Clone of TaHKT8 gene Primers were designed according to wheat TaHKT8 registered in NCBI. We obtained a band of about 1600bp by RT-PCR amplification and sequencing result was consistent with the original sequence.2. Analysis of TaHKT8 structure By using a variety of bioinformatics technology, the structure of nucleotide and amino acid sequence of wheat TaHKT8 were analyzed. The sequence located at 196~1590bp in mRNA of TaHKT8 is a single exon region, i.e. open reading frame (0RF), but the poly A signal is not obvious. The molecular formula of TaHKT8 protein is C2618H4096N678O712S26, relative molecular weight 57.3kD, isoelectric point 8.90, theoretical half-life less than 30h, instability parameter 29.75, belonging to the stable protein. Prediction of secondary structure indicates that, TaHKT8 is composed of 42.05%α-helix, 18.80%β-extended chain, 5.81%β-turn and 33.33% random coil.α-helix and random coil are mainly structural elements, andβ-turn and extended chain locate throughout the protein. TaHKT8 contains 10 transmembrane regions, N-side is located in the outer membrane, C-side in the membrane, 175 of the amino acid residues are outside of the membrane, accounting for 33.9%, the number of amino acid residues that in membrane is 341, about 66.1%. The homologous alignment shows that TaHKT8 gene is highly conservation in different plants. The construction of the phylogenetic tree shows that sequence of TaHKT8 gene is closely related to OsHKT8(rice),TaHKT7 (wheat) and OsHKT7(rice) which has salt-tolerant functions. Analysis structure and function domain shows that TaHKT8 protein contains TrkH domain which has cation transport function and can transport K+. Protein With TrkH domain can consume ATP to transport sodium ions in order to maintain a high K+/Na+ ratios, reducing the damage caused by high concentration of Na+ in cytoplasm. 3. Test TaHKT8 function Eukaryotic protein expression technology was used to verify salt-tolerance function of TaHKT8. A yeast expression vector for TaHKT8 gene, TaHKT8-pYES2, was constructed. The plasmid constructed was transformed with yeast cells AXT3K by method of PEG and heat stimulation. Selection by medium of YNB+A+T, transgenic yeast cells were gotten. Results identified by PCR, restriction enzyme digestion and sequencing showed that TaHKT8 gene was integrated into the genome of yeast cells. In the AP medium containing 30~40mM NaCl, the growth of wide type yeast cells was significantly inhibited, while transgenic cells grew normally. Results indicated that product of TaHKT8 gene might have made yeast cells strong salt-tolerant capacity. In different KCl concentrations, however, there were no differences between transgenic and WT cells and both of cells grew well on the 0.5M KCl and dead on 0.6M KCl medium. It was suggested that the target gene could alleviate the poisoning effect of sodium rather than potassium ions.
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