Potassium Transporters TaHak17 And TaHak4 In Wheat Functional Verification Of Genes

Among the plant potassium transporter families,HAK/KUP/KT gene family has the largest number of members,which can promote plant growth and improve plant adaptability to the environment.Ta HAK17 and Ta HAK4 are located in the fourth cluster of HAK/KUP/KT gene family,and the functions of Ta HAK17 and Ta HAK4 are verified in this experiment.The main research results are as follows :(1)K~+ transport function of Ta HAK17 was identified by yeast heterologous expression system.The yeast mutant CY162 transformed with Ta HAK1 gene was used as the positive control,and the CY162 transformed with empty vector p416 was used as the negative control for yeast functional complementation verification.The results showed that the Ta HAK17 control strain and the p416 yeast strain could grow normally under the condition of normal K~+ concentration(100 m M).Under the low potassium stress with K~+ concentration of 1 m M,the p416 empty vector-transformed strain could not grow normally,and the Ta HAK17 and Ta HAK1 positive control strains could grow normally,indicating that Ta HAK17 could complement the K~+transport function of yeast mutants.Under the condition of normal potassium ion concentration,K~+ concentration of 100 m M,with the increase of Na+ concentration,the yeast strains transformed with Ta HAK17,Ta HAK1 and empty vector p416 could grow normally.When the concentration of K~+ was 1 m M under low potassium stress,with the increase of Na+ concentration,the yeast strains transformed with Ta HAK17 and empty vector p416 could not grow normally,while the positive control strain transformed with Ta HAK1 could survive,indicating that Ta HAK17 and Ta HAK1 have different functions.Ta HAK17 only has low affinity K~+ absorption function and does not have salt tolerance under low potassium stress.The results of yeast growth curve and K~+ content in yeast showed that the yeast transformed with Ta HAK17 and the yeast transformed with empty vector p416 could grow normally at K~+ concentration of 100 m M.When the K~+ concentration was 1 m M,the yeast transformed with p416 empty vector could not grow,while the yeast transformed with Ta HAK17-p416 could grow normally,further indicating that Ta HAK17 had low potassium transport function.(2)Under low potassium stress,Ta HAK17 can partially compensate for the phenotypic defects of Arabidopsis athak5 single mutant and atakt1/athak5 double mutant under low potassium stress.In order to further verify the low potassium transport function of Ta HAK17 in plants,the root tip K~+ flow rate of Ta HAK17 transgenic A.thaliana and the control was determined by non-invasive microassay.The K~+ flow velocity in different parts of the root tip of Columbia wild-type Arabidopsis seedlings grown for 10 days was measured.It was found that the K~+efflux rate in the apical meristem area was the largest,followed by the root cap and the elongation area.The K~+ flow rate in apical meristem was measured for Ta HAK17 transgenic Arabidopsis athak5 single mutant,Arabidopsis atakt1/athak5 double mutant and Colombian wild-type Arabidopsis.The results showed that the K~+ efflux rate of athak5 single mutant and atakt1/athak5 double mutant of Ta HAK17 was higher than that of non-transgenic control in different concentrations of test solution,while the K~+ efflux rate of wild-type Colombia transgenic Ta HAK17 was the same as that of control.The results showed that Ta HAK17 gene could promote K~+ transport in plant roots.(3)Ta HAK4 gene was cloned by homologous cloning,and the K~+ transport function of Ta HAK4 was identified by yeast heterologous expression system.Through yeast functional complementation verification,yeast growth curve determination,and K~+ content determination in yeast,it was found that Ta HAK4 could not grow normally when K~+ concentration was lower than 1 m M,and could grow normally when K~+concentration was higher than 1 m M,that is,Ta HAK4 could not promote K~+ transport under low potassium conditions.Subcellular localization of Ta HAK4 in wheat protoplast was detected,and the results showed that Ta HAK4 was expressed in cell membrane,nucleus and chloroplast.