Cloning And Expression Pattern Analysis Of Major High-Affinity K~+ Transporter Genes From Halophyte Puccinellia Tenuiflora

Increasing soil salinity (especially in the cultivated land) is a serious threat to plant growth and agricultural productions all over the world. The dominant ion in the saline soils is Na+ which can cause ion toxicity and osmotic stress and interrupt the normal process of metabolism. In the long-range evolutionary process, a number of plants have been developing some kinds of mechanisms to accommodate to salt stress. For wheat and Puccinellia tenuiflora, they resist salt stress by means of Na+ exclusion. Due to living in the affluent environment chronically, wheat has the limited ability of Na+ exclusion; By contrast, Puccinellia tenuiflora whose body contains many genes related to salt tolerance has the much stronger ability to exclude Na+ and uptake K+ alternatively. Many studies have proved high affinity K+ transporters play important roles in restricting Na+ uptake and in absorbing K+ alternatively to transport to shoot. Therefore, cloning HKT genes from Puccinellia tenuiflora will have a positive effect on our next-step work.In this study, PutHKT2;1, PutHKT1;5 and the fragment of PutHKT1;4 were cloned from Puccinellia tenuiflora by means of RT-PCR and RACE; Then PutHKT2;1 plant expression vector was constructed based on the vector PBI 121; Taking Actin as a internal control, we analyzed the expression pattern of PutHKT2;1 and PutHKT1;5. The results are as follows:1. The PutHKT2:1 cDNA is 1919 bp and contains an ORF of 1641 bp encoding 546 amino acid residues,3’UTR of 260 bp and 5’UTR of 18 bp. PI is 9.07 and Mw is 60.5 kDa. The BLAST data showed PutHKT2;1 shares 66% homology with HKT2;1 of other plants. It may have 11 transmembrane segments. Analysis of the PutHKT2;1 secondary structure showed it contains 47.99% α-helix,5.13% β-turn,31.87% coil and 15.01% extended strand.2. The PutHKT1;5 cDNA is 1810 bp and contains an ORF of 1587 bp encoding 528 amino acid residues,3’UTR of 174 bp and 5’UTR of 46 bp. PI is 8.86 and Mw is 58.3 kDa. The BLAST data showed PutHKT1;5 shares 62% homology with HKT1;5 of other plants. It may have 8 transmembrane segments. Analysis of the PutHKT1;5 secondary structure showed it contains 43.29% α-helix,6.24% β-turn,32.51% coil and 17.96% extended strand.3. The fragment of PutHKT1;4 is 629 bp encoding 209 amino acid residues. The BLAST data showed PutHKTl;4 shares 61% homology with HKT1;4 of other plants. It may have 5 transmembrane segments.4. Along with the increase of Na+ concentration, the Na+ content shows a upward tendency while the K+ content decreases sharply. The increasing ST shows the ability of excluding Na+ and uptaking K+ is much stronger under NaCl stress.5. The expression of PutHKT2;1 was induced dramatically in roots under K+-starvation, but its expression level was down-regulated when NaCl was added. It may play an important role in the high affinity K+ uptake and in the low affinity Na+ uptake in K+-starvation condition. However, PutHKT1;5 shows a different expression pattern. It was induced under high salt stress and its expression level is not affected by K+ concentration, suggesting that it may exclude Na+ when Puccinellia tenuiflora is under salt stress. To sum up, along with the increase of Na+ concentration, ST is increasing due to the PutHKT2;l and PutHKT1;5 harmonization in root and shoot of Puccinellia tenuiflora.