Cloning And Functional Characterization Of TaAKT1

K+ is the most abundant cation in plant cell,consisting of more than 2%of dry weight.Due to its flowability in plants,K+ is a major osmotic element,playing an essential role in the process of plant growth and development as well as stress-resisting.In order for plants to grow well,a variety of K+ transporters are needed to maintain their K+ homeostasis by cooperate with each other to mediate K+uptake and transport as the result of the fact that concentration of K+ in soil is only 1%of that in plant and different organs or tissue may need different concentration of K+.Wheat is one of the most important crops in China and K+-deficiency has a bad influence in its yield and quality,which also weakens its stress-tolerance.Most of cultivated land in China lacks K+,so it is significant to reveal the mechanism of potassium uptake in wheat.Recent study has elucidated the K+-uptake mechanism in the model plant Arabidopsis thalzana:in low-K+ condition,AtAKT1 is a predominate potassium transporter to mediate K+ uptake from soil to root,and it is activated by AtCIPK23.In this study,we cloned TaAKT1 and TaCIPK23 from wheat and conducted a complementally functional experiment by using the Saccharomyces cerevisiae mutant WA3 to identify the function of TaAKT1 and unravel the regulatory mechanism in K+ transport.1.Homologous cloning methods were employed to get TaAKT1 and TaCIPK23 genes.The deduced open reading frame of the TaAKT1 gene was 2694 bp encoded a protein of 897 amino acid residue.The predicted molecular mass of TaAKT1 was 100.92 kD and isoelectric point of 7.63,while TaCIPK23 gene had a coding region of 1362 bp,which translated into 454 amino acid.The results of bio-information analysis suggested that TaAKT1 shares highly identity 97.22%,86.5%,76.03%and 60.55%in amino acid sequences with HvAKT1,BdAKT1,OsAKTl and OsAKT1 respctively.2.The RT-PCR result indicated that in K+-deficiency condition,the expression level of TaAKT1 was up-regulated in wheat root while that of TaAKT1 in wheat leaf did not change dramatically.The result showed that TaAKT1 prefers to expressed in specific tissue.3.TaAKTl was transformed into the yeast mutant strain WA3,which could not grown normally on the AP medium added less than 10 mM KCl because of lacking the endogenous high-affinity K+-uptake transporters TRK1 and TRK2,to analyze its function.W?3 transformed empty vector could not grow normally on the AP medium added less than 10 mM KCl while W?3 transformed TaAKT1 could.This suggested that TaAKTl might mediate K+ uptake.4.We transformed both TaAKT1 and TaCIPK23 into W?3.W?3 transformed both TaAKT1 and TaCIPK23 could even grow on the AP medium added extremely low KCl(60 ?M).By contrast W?3 only transformed TaAKT1 could not grow on the AP medium added 60 ?M KCl.We deduced that TaCIPK23 can activate TaAKT1 and enhance its ability of mediating K+ uptake.In summary,we cloned TaAKT1 and TaCIPK23 from wheat and analyzed TaAKT1 protein by bio-information method.TaAKT1 was mainly expressed in root,and under low-potassium condition the expressional level of TaAKT1 in root was up-regulated,while that in leaf did not change significantly.Based on functional expression in yeast,TaAKT1 was thought to candidate as a potassium transporter and its ability was enhanced by TaCIPK23.