Cloning And Functional Analysis Of A High Affinity K~+ Transporter Gene SeHAK1 From Salicornia Europaea

Salinity and deficiency of K+ in the soil affected plant growth and development. Under these dual stresses, it is essential to maintain the ion homeostasis and to enhance the ability of high salt and low [K+] tolerance for plants. High affinity K+ transporter is a contributor to conduct K+ uptake. Under high salt condition, Na+ sensitive high affinity potassium transporter may be affected, while Na+ insensitive high affinity K+ transporter could be less affected, which may be an essential protein to transport K+ and to enhance salt tolerance of plants under low [K+] and high [Na+].Some Na+ insensitive K+ transporter have been cloned from halophytes. Salicornia europaea is one of halophytes that grow at the extreme salinities. We hope to obtain SeHAK1 from Salicornia europaea and it would be a Na+ insensitive potassium transporter not only contributing the K+ uptake but also enhancing salt tolerance of plants.SeHAK1 encoding a Na+ -insensitive K+ transporter was isolated from Salicorina europaea by RT-PCR, RLM-RACE and Anchor PCR technique. The Sequence analysis of SeHAK1 indicated that it belongs to groupⅡof HAK/KUP/KT. The cDNA of SeHAK1 (2706bp) contained 249bp 5'UTR,96bp 3'UTR and 2361bp ORF sequence, which encodes 786 amino acids that showed high homology from 40% to 77% with other high affinity K+ transporter. The hydrophobic analysis indicated SeHAK1 has 14 transmembrane domains.The expression analysis of SeHAK1 indicated that SeHAK1 was expressed at root and shoot tissues of S. europaea. SeHAKl was detected under high salt treatment, K+ -starved and non-treatment conditions.The ORF of SeHAK1 was constructed into the pYES2.0 and the pYES-SeHAK1 was transferred into the trk1 trk2 mutant yeast WΔ3 by electroporation method for yeast expression. The WΔ3-SeHAK1 could grow at 100μM K+ medium and was suppressed by Cs+ and Rb+ but less affected by high [Na+]. Functional analysis of SeHAK1 in yeast mutant WΔ3 indicated that SeHAK1 conduct high affinity potassium uptake with Km of 3.8μM. Under lOmM External [K+] condition, the K+ uptake ratio of WΔ3-SeHAK1 was higher than WΔ3, which indicatied that SeHAKl also media low affinity potassium uptake.