Functional Analysis Of The HKT Gene From Thellungiella Parvula

The plant HKT(high-affinity K~+ transporter)is a Na~+/K~+ transporter or Na~+/K~+ co-transporter.HKT plays an important role in plant salt tolerance.The Arabidopsis At HKT1;1,which is localized in the plasma membrane of the phloem parenchyma cells,is critical for Na~+ efflux from leaves,avoiding the harmful effects of salt stress.Thellungiella parvula belongs to the family Crucifera.It is closely related to Arabidopsis thaliana,and is highly tolerance to salt stress.Thus,T.parvula can be used as a model for studying the mechanism of salt tolerance.T.parvula has two HKT genes: TpHKT1;1 and TpHK1;2.However,their ion transport activity and function remain unclear.We aim to understand the ion transport properties of HKT genes and their function in salt tolerance.Using molecular genetic methods.we cloned TpHKT1;1 and TpHK1;2 genes,and expressed them in Arabidopsis and yeast(Saccharomyces cerevisiae),We examined their expression patterns and subcellular localizations.We also analyzed their ion transport properties and physiological functions.The major results are listed below:1.We expressed TpHKT1;1 and TpHK1;2 genes in Arabidopsis wild type Col(gl1)and athkt1-1 mutant to obtain the transgenic plants.2.The tissue expression patterns of TpHKT1;1 and TpHK1;2 genes were examined by GUS staining.TpHTK1;1 was mainly expressed in leaf tips and the stele of roots,but was weak in the silique tip and fruit stalk,as well as the stamen and stigma;TpHKT1;2 was significantly expressed in leaves and hypocotyls,but was weak in lateral root primordia and siliques.TpHKT1;2 was strongly expressed in the stamen and stigma compared to TpHTK1;1.These results suggest that TpHKT1;1 and TpHKT1;2 have similar expression patterns but difference exists.3.To examine the subcellular localization of TpHKT1;1.The GFP-TpHKT1;1/Col(gl1)transgenic plants were hybridized with Arabidopsis plants expressing organelle markers to prepare the double co-expression plants.we found that TpHKT1;1 was localized in the plasma membrane,Golgi,trans-Golgi networks(TGN),and prevacuolar compartment(PVC)/multivesicular bodies(MVB),indicating that TpHTK1;1 is widely expressed in plants.4.Expression of TpHKT1;1 and TpHK1;2 genes in the yeast AXT3 mutant enhanced yeast resistance to the stress of hygromycin B,NaCl and LiCl,indicating that TpHKT1;1 and TpHK1;2 mediate Na~+ and Li~+ transport,and involve in vesicle trafficking.5.We expressed the TpHKT1;1 and TpHK1;2 genes in Col(gl1)and athkt1-1 to obtain the transgenic plants.We found that the transgenic plants were inhibited in seed germination and seedling growth under NaCl treatment.When growing in soil under 200 m M NaCl treatment,the growth of the transgenic plants was significantly inhibited,indicating that TpHKT1;1 and TpHK1;2 genes are functioning in moving Na~+ into the cell.Also,the transgenic plants were sensitive to LiCl stress;expression of TpHKT1;2 was more sensitive than the expression of TpHKT1;1.In conclusion,TpHK1;1 and TpHK1;2 function in moving Na~+ into the cell;TpHKT1;1 and TpHKT1;2 have different transport molds for Na~+ and Li~+.