Function And Regulation Of Potassium Channel Gene NTORK1 In Tobacco Root

For the study of tobacco potassium channel NTORK1 whether regulated by plant hormones involved in the regulation of root potassium efflux, a plant expression vector containing promoter of NTORK1 and a plant expression vector containing RNAi fragment of NTORK1 were constructed by using p ER8 and p BI121 as the material. Using Agrobacterium transformation and transient transformation, we research the function of the promoter of NTORK1 and the function of NTORK1. Through the study we obtained the following results:1. Cultivating tobacco K326 plants under the condition of low potassium hydroponic,treating plants with different concentrations of SA and NAA, detected the root extraradical potassium content. At the same time, using Real-time PCR analyzed the expression of NTORK1. The experimental results showed that root K+ efflux could be inhibited by the medium of 50μmol/L SA. However, the solution of 500μmol/L SA could induce the root and K+ efflux and could induce the expression of NTORK1.2. Constructed the p BI121-NTORK expression vector which was based on the vector p BI121 vector, where taken place the 35 S promotor with the sequence cloned from a fragment of 2111 bp upstream the NTORK1. Then, the plant expression vector of p BI121-NTORK was constructed with NTROK1:GUS structure.3. The expression vector p BI121-NTORK was introduced into tobacco K326 leaf by transient expression. The results showed that the NTORK1 promoter was induced by 500μmol/L SA and 50μmol/L NAA.4. Designed and constructed NTORK1 gene RNAi expression vector. The vector was based on the p ER8 vector, containing a RNAi fragment designed for NTORK1, Tnos terminator, DR5 promoter and GUS cloned from the vector p BI121. Among them, the RNAi fragment was derived from the NTORK1 gene 3’- terminal 250 bp and actin intron.5. The NTORK1 gene RNAi expression vector was introduced into tobacco K326 leaf by Agrobacterium-mediated transformation. Multiple transgenic tobacco plants were obtained by PCR identification. Transgenic tobacco plant was induced by 5 μmol/L 17-β-Estradiol nutrient solution. Analyzed by Real-time PCR to examine the function of RNAi fragment. The results showed that the RNAi could significantly reduce the NTORK1 expression, the inhibition rate reach to 90%.6. Transgenic tobacco plant and non-transgenic K326 plant cultivated 3d under the condition of 5 μmol/L 17-β-Estradiol nutrient solution hydroponic. Successively cultivated in 5 μmol/L Es, 5 μmol/L Es + 50 μmol/L SA, 5 μmol/L Es + 500 μmol/L SA Hoagland medium 40 min respectively. The potassium concentration of solution was detected and the NTORK1 expression was detected by Real-time PCR. The results showed that transgenic and non-transgenic K326 plants NTORK1 expression increased compared to low concentration of SA in the condition of high concentration of SA. At the same time, the transgenic plants potassium efflux was significantly lower than non-transgenic K326 plants in high concentration of SA. Ultimately concluded, NTORK1 mediated potassium efflux in roots and regulated by SA, while it was inhibited by 50 μmol/L SA and was induced by 500 μmol/L SAFinally, the obtained results were as followed:1. NTORK1 expression could be induced by high concentration of SA and low concentration of NAA; 2. high concentration of SA could induce root potassium efflux,while low concentration of SA could inhibit the root potassium efflux;3. NTORK1 mediated potassium efflux and regulated by SA. This study combined plant hormones, NTORK1 and root potassium efflux, providing a theoretical basis to improve the potasium content of tobacco.