Functional identification of tonoplast membrane potassium channel gene NtTPK in tobacco
The presence of K+ is crucial for maintaining the quality of tobacco, and hence the study related to understanding the molecular regulation of the concentration of K+ in tobacco could be of great significance to improve the quality of tobacco. In this study, a two-pore K (TPK) channel gene NtTPKa was cloned from tobacco. The gene was found to encode a protein containing the unique K+ selection motif GYGD and the transmembrane region of TPK1. The expression of NtTPKa gene was substantially increased by low potassium stress. In addition, analysis of subcellular localization showed that NtTPKa was primarily localized in the tonoplast membrane. The concentration of K+ in tobacco was significantly increased by application of different materials that inhibited the expression of NtTPKa gene by RNAi. We used CRISPR/Cas9 technology to create NtTPKa gene knockout materials, and the concentration of K+ in tobacco was observed to be significantly increased. Therefore, NtTPKa could serve as a negative regulatory gene for concentration of K+ in tobacco. We also examined the K+ transport function of NtTPKa channel protein through patch clamp technique. The results showed that the NtTPKa protein only selectively transported K+, but possessed no transport function for Na+, Mg2+ and Ca2+, and its transport activity for K+ exhibited specific concentration of K+ dependence. The clone identification of NtTPKa can potentially provide a new gene resource for increasing the concentration of K+ in tobacco.