Analysis Of Na~+ Transport Activity Of The HKTs From Thellungiella Parvula

PlantHKTs(high affinity K~+ transporters)play an important role in salt tolerance in plants.WheatHKT gene,Ta HKT2;1,is the firstHKT gene to be cloned in plants.Ta HKT2;1 has been identified to be a high affinity Na~+/K~+ co-transporter.Arabidopsis has only one HKT gene,namely AtHKT1;1.Studies showed that AtHKT1;1 had Na~+ transport activity.Thellungiella parvula belongs to the family Crucifera.It is closely related to Arabidopsis thaliana,and is highly tolerance to salt stress.Thus,identifying the salt tolerant genes from Thellungiella parvula is helpful for revealing the molecular mechanism underlying salt tolerance in plants.Thellungiella parvula has two HKT genes(TpHKT1;1 and TpHK1;2).However,their ion transport activity and functions remain unclear.In order to understand the function of the HKT genes from Thellungiella parvula,we cloned TpHKT1;1 and TpHK1;2 genes,and expressed them in Arabidopsis and yeast(Saccharomyces cerevisiae).We analyzed the ion transport activity and function of TpHKT1;1 and TpHK1;2.Meanwhile,we observed the subcellular localization of TpHKT1;2 using gene expression techniques.The major results are listed below:1.We cloned TpHKT1;1 and TpHK1;2 genes.We then expressed them in Arabidopsis wild type Col(gl1)as well as athkt1-1 and athkt1-4 mutants.We have obtained the TpHKT1;2transgenetic plants.We also cloned TpHKT1;1 and TpHK1;2 to yeast expression vector p DR196,and expressed them in yeast.2.We found that TpHKT1;2 transgenetic plants stopped growing and were dead under the treatment of 50 m M Na Cl,while the wild type Col(gl1),athkt1-1 and athkt1-4 grew well.This results indicates that TpHKT1;2 may have the inward Na~+ transport activity.3.Using yeast expression assay,we found that the salt sensitive yeast mutants expressing TpHKT1;1 and TpHKT1;2 did not grow under 30 m M Na Cl;However,these yeast strains conferred salt tolerance in the presence of 1 m M or 7m M KCl.These results indicate that TpHKT1;1 and TpHKT1;2 have Na~+ and K~+ transport activity;K~+ inhibits the Na~+ transport activity of TpHKT1;1 and TpHKT1;2.4.To analyze the subcellular localization of TpHKT1;2,we co-expressed TpHKT1;2 and organelle markers in Arabidopsis to prepare transgenetic plants.Using confocal microscopy,we found that TpHKT1;2 was localized in the plasma membrane,Golgi,trans-Golgi networks and multivesicular bodies.In conclusion,TpHK1;1 and TpHK1;2 have inward Na~+ transport activity;K~+ can inhibit the Na~+ transport activity of TpHK1;1 and TpHK1;2.