The Subcellular Localization And Function Research Of High-affinity Potassium Transporter ZmHAK1 In Maize

Potassium is one of the essential nutrient elements with important physiological functions in plants and can effectively increase the yield and quality of crops.However,due to the leaching loss of potassium in soil,the shortage of potassium in cultivated land has become an important factor restricting agricultural production in China and even in the world.Because of the shortage of potassic fertilizer supply,it is an effective measure to cope with the shortage of potassium in cultivated land by discovering the plant resources that can highly utilize potassium and using the genes of highly utilizing potassium in these plants to increase the yield and quality of plants in a low amount potassium soil.According to reports in literatures,plants mainly absorb K⁺from environment through low-affinity potassium channel proteins and high-affinity potassium transporter proteins.High-affinity potassium transporters have the function of promoting K⁺uptake in a low amount potassium stress.The KUP/HAK/KT family is the largest family of high-affinity potassium transporter proteins in plants.Our research group had cloned ZmHAK1 gene from maize which could highly utilize potassium.Bioinformatics analysis indicated that this gene belonged to the KUP/HAK/KT family.The purpose of this study was to identify the specific expression site of ZmHAK1 gene in cells by subcellular localization technology,and to study the function of this gene by RNA interference technology.In this study,Agrobacterium was used to infect the leaves of N.benthamiana,and subcellular localization of ZmHAK1 gene was studied by observing EYFP fusion protein.The results showed that ZmHAK1 was mainly located on the plasma membrane,and this result was same as the function of this protein and the prediction result of software.The constructed pRNAi-ZmHAK1 RNA interference vector was transformed into Arabidopsis thaliana strains which containing ZmHAK1 gene by flower infection.Then transgenic strains of T₃ generation were gotten through screening and selfing.The transgenic maize with pRNAi-ZmHAK1 RNA interference vector was also gotten by pollen-tube pathway.QRT-PCR analysis was performed with T₃ RNA interference plants.The results showed that the expression level of ZmHAK1 was inhibited under a low amount potassium stress,and the up-regulation of ZmHAK1 in RNA interference strains were significantly lower than that of the Arabidopsis ZmHAK1 functional recovery line.The expression level of ZmHAK1 was highest at 72h and then began to decline;The expression of ZmHAK1 in roots was higher than that of stems and leaves.The results of growth potential and potassium uptake indicated that the growth of RNA interference strains was inhibited under a low amount potassium stress,and the root elongation of Arabidopsis in this strain was significantly inhibited when the K⁺was less than 100?mol/L,but the root elongation was hardly affected when the K⁺was more than 200?mol/L.From 15 days under low amount potassium stress,significant symptoms of potassium deficiency were observed in RNA interference strains compared with the control.Furthermore,the potassium uptake and potassium utilization index of RNA interference strains under a low amount potassium stress were lower than that of control.