| The salt overly sensitive(SOS)pathway plays an essential role in maintaining ion homeostasis and conferring salt tolerance in plant.SOS pathway consists of three proteins including plasma membrane Na+/H+ antiporter SOS1,protein kinase SOS2 and Ca2+-binding protein SOS3.SOS1 is an important factor for plant salt stress which can excrete excess Na+ from plant cell under salt stress.In normal condition the function of SOS1 is unactivie,while SOS2/SOS3 complexes kinase can active the function of SOS 1.Here we identified two SOS components in wheat(Triticum aestivum),designated as TaSOS2 and TaSOS3 by blast analysis.We study the mechanism of wheat SOS pathway using technique of yeast mutants AXT3K,Arabidopsis mutants sosl and yeast two-hybrid system.The main results are as following:1.We cloned TaSOS2 and TaSOS3 which are the components of wheat SOS pathway.Homology comparison showed that TaSOS3 has 60.81%identity with Arabidopsis AtSOS3 and the 81.65%identity with rice OsCBL4(OsSOS3).TaSOS2 and AtSOS2 protein sequence homology is 68.08%.TaSOS2 and the rice OsCIPK24(OsSOS2)homology is 86.83%.2.TaSOS3,TaSOS2 and TaSOSl are transformed into yeast mutant strain AXT3K.The phenotype of salt resistant was detected by drop test of tansgenitic yeast cell on AP medium added different density of NaCl.Yeast mutant AXT3K lacking endogenous salt tolerant gene the which the growth was inhibited and cannot survive on the AP medium with the addition of 40mM NaCl.But the AXT3K transformed with TaSOSl can grown on the AP medium with 80 mM NaCl.TaSOS2 can activate TaSOSl activity,can growth in the AP with 400 mM NaCl,while TaSOS3/TaSOS2 protein kinase complex and TaSOSl coexpression also can growth at 400 mM NaCl AP medium.3.Bioinformatics analysis showed that TaSOSl gene encodes a protein with 1142 amino acid residues and its N-terminal containing 12 transmembrane regions,the hydrophilic C-terminal encoded 713 amino acid residues which distributed in the cytoplasm.To determine the binding site of TaSOS2 to TaSOS1,C-terminal deletion mutants of TaSOS1 was constructed by delete amino acid residues every 20 amino acid residues from C-terminal,the mutant named Δ1122,Δ1102,Δ1082,Δ1062,Δ1042,Δ1022,Δ1002 respectively according to the residues of amino acid.The mutants are coexpressed with TaSOS2 or TaSOS2/TaSOS3 complex in yeast strain AXT3K.The result show that TaSOS2 can’t regulate the function of Δ1122 and the mutants shorter thanΔ1122,but TaSOS2/TaSOS3 complex can active the function of Δ1122.This indicated that the binding site of TaSOS2 to TaSOSl was in the residues after 1122,but TaSOS2/TaSOS3 composite binding site was in the site of residues before 1122.TaSOS2 and TaSOS2/TaSOS3 complex have different binding sites to TaSOSl.To determine TaSOS2 binding site in TaSOS1,the TaSOS1 deletion mutation Δ1132 was constructed.Yeast complementation test results showed that TaSOS2 can regulate Δ1132.Therefore,the locus of TaSOS2 binding to TaSOS1 in the 10 amino acid residues between 1122 to 1132,may be serine 1126 and serine 1128.Point mutations were designed as 1126A/1128D by using 1126S mutant to A and 1128S mutant to D.Yeast complementation test showed that the point mutation can’t regulate by TaSOS2,these results demonstrated that TaSOS2 binding to 1126S and 1128S of TaSOS1.4.TaSOSl and Δ1122 were transformed to Arabidopsis mutant sos1,results showed that the transfer Δ1122 has the same phenotype with the plant that transferred with the wild type of TaSOS1.The result demonstrated that Δ1122 can interact with SOS2/SOS3 complex of Arabidopsis mutant sosl and can enhance plant salt tolerance.The binding site of TaSOS2/TaSOS3 complex and TaSOSl was in the residue of before the 1122,which was different from the bonding site TaSOS2 to TaSOS1.5.In order to identify the inhibition domain of TaSOS1 and get the mutant with higher salt tolerant,we performed the C-terminal deletion mutants Δ428,Δ5329,Δ697,Δ727,Δ974.These mutants were transformed to yeast strain of AXT3K respectively by PEG transformed method and the yeast complementation tests were assay.The function of salt tolerance was lost in the mutants of Δ428 which lack of complete C-terminal,Δ532 and Δ697 which lack of lager C-terminal of TaSOS1.The salt tolerance function of muntants which got after Δ727 have the better phenotype than TaSOSl.Among the mutants the Δ974 has the best phenotype of salt tolerance.When the trunctated Δ974 was expressed in AXT3K cells,halotolerance was completely recovered and cells could grow in AP midum supplied up to 1M NaCl.6.The higher activity mutant Δ974 was transformed to Arabidopsis thaliana and the ion transport activity of Na+ and K+ were measured using MIFE technique.The result proved that Δ974 has higher salt-tolerant activity because Δ974 has the faster Na+ efflux speed than the wild type of TaSOS1 at the same condition.Δ974 is of higher salt tolerance and can enrich the wheat genetic resources and which can be an important gene for wheat salt tolerant genetic engineering. |
