Cloning And Characterization Of A K~+ Transporter Gene TrkH From Marine Microorganism

Potassium is an essential nutrient and the most abundant cation widely distributed in cells and organs, which participates in many vital physiological process. A high and relatively stable K+ concentration in certain cells is important. In contrast, Na+ is toxic at high concentration in cytosol. In China, potassium deficiency is one of the most important factors that restrict agriculture production, and potassium deficiency is often associated with a certain degree of salinization. So we aim at finding a Na+ insensitive K+ transporter which can maintain K+ uptake under salt stress, in order to improve the abilities of potassium absorption in transgenic plants under high-salt conditions. Although several genes of different potassium transporters or channels have been characterized from various plants, most of them come from glycophyte and sensitive to salt stress. Some K+ transporters of microorganism from halobiotic environment have been shown to increase K+ absorption under salt stress. The marine environment is rich in Na+, and we speculate that the Na+ insensitive K+ transporter can be obtained from microorganisms in the ocean. Since more than 99% of microorganisms in ocean environment are unculturable, so we choose marine microbial metagenomics DNA as the experimental materials. In this study, a K+ transporter gene trkH was cloned from marine microbial metagenomics DNA, which contained an open read frame of 1,392 bp encoding 463 amino acids. Sequence analysis predicted that the peotein contained ten trans-membrane domains, and belonged to the TrkH superfamily, which is a typical K+ transporter family in bacteria. We performed complementation tests using a K+ uptake-deficient mutant of yeast strain CY162, and found that TrkH can rescue the growth and K+ uptake deficiency of CY162 under 3mM K+ conditions. When the medium contain 3mM K+ and 1 OOmM Na+, the yeast cells expressing TrkH showed some degree of sodium insensitivity compared with the cells expressing EcTrkH of E. coli. It is the first time to comfirm the bacterial TrkH’s character in eukaryotic system. To further investigated the function of TrkH in plants, we established transgenic Arabidopsis thaliana and screened the T1 generation of Arabidopsis with Basta.