Gene Cloning And Functional Verification Of Wheat Potassium Transporter Gene TaHAK5

Potassium is one of the essential elements required for plant growth and development,and is an important element of stress resistance and quality.It is one of the effective measures to mitigation soil potassium deficiency by excavating the genes for efficient potassium ion utilization in plants.Under K+deficiency,high-affinity K+transporters could promote the ability of plants to absorb K+.The HAK/KUP/KT family is the largest family of high-affinity potassium transporter proteins.In this study,we analyzed the expression level of TaHAK5 gene by quantitative real-time PCR;determined the site of gene expression products by subcellular localization techniques;preliminarily identified the potassium transport function of TaHAK5 gene by yeast complementation assay;and transferred TaHAK5 into wild-type Arabidopsis for overexpression to verify the function of TaHAK5 gene in plants.The main test results are as follows:1.The specific expression of TaHAK5 gene in different tissues of wheat was analyzed by Real-time PCR.It was found that TaHAK5 gene was expressed in all tissues of wheat plants,with the highest expression level in young leaves,followed by stem,old leaves and roots,and the lowest expression level in grains.This indicates that TaHAK5 gene is not tissue-specific in wheat plants,which not only participates in the potassium ion transport function in plant roots,but may also regulate plant growth and development.2.Real-time PCR was used to analyze the expression of TaHAK5 gene in wheat roots and leaves under K+stress.The results showed that the expression of TaHAK5 gene was up-regulated significantly in wheat roots under K+ stress,and the expression of TaHAK5 gene reached a peak at 12 h of stress treatment,which was about 11 times higher than that under non-stress treatment.TaHAK5 leaf expression was down-regulated and peaked at 96 h,about 1.7 times higher than that under unstressed treatment.TaHAK1 gene expression was up-regulated in both roots and leaves.3.Subcellular localization experiments showed that TaHAK5 gene was mainly localized on the cell membrane,indicating that TaHAK5 gene may function on the cell membrane.4.TaHAK5 was transferred into the potassium-sensitive yeast mutant CY162,and the results showed that TaHAK5 gene could restore the defective phenotype of CY162 and could grow normally on low-potassium medium;it was found in the growth curve in yeast,potassium ion depletion experiment,and intracellular potassium ion content assay experiment in yeast,while under low-potassium conditions,the growth rate and growth amount,absorption rate and uptake amount of external potassium ions,and intracellular potassium ion content of the yeast strain transfected with TaHAK5 gene were higher than those with TaHAK1 gene,indicating that TaHAK5 gene has the ability to transport potassium ions,and TaHAK5 was also found to transport potassium ions more than TaHAK1 gene.5.The TaHAK5 gene was transferred into the model crop Arabidopsis.The results showed that the growth of transgenic Arabidopsis was better than that of wild type and the root length was longer than that of wild type Arabidopsis on low potassium medium.At 0.1 m M K+,the root length of TaHAK5 transgenic Arabidopsis was about 2.5 times longer than that of wild type Arabidopsis,and that of TaHAK1 transgenic Arabidopsis was about 1.7 times longer than that of wild type Arabidopsis.At 0.1 m M K+,the root length of TaHAK5 transgenic Arabidopsis was about 1.4times longer than that of TaHAK1 transgenic Arabidopsis,and the fresh weight of TaHAK5 transgenic Arabidopsis was about 1.19 times longer than that of TaHAK1 transgenic Arabidopsis.Showed that TaHAK5 gene could promote the elongation of plant roots and participate in K+uptake in roots,and TaHAK5 was involved in better K + uptake ability in roots than TaHAK1 gene.