Functional Analysis Of The Potassium Transporter Gene TaHAK13 In Wheat

K+is an essential nutrient element for plant growth and development.HAK/KUP/KT is the main potassium ion transporter in plants,which participates in the absorption and transportation of K+by plants and plays an important role in the absorption and utilization of potassium nutrition by plants.The functions of HAK/KUP/KT family genes have been identified in model plants,but there are few reports on the function of this family gene in wheat.In this study,TaHAK13 gene was cloned from wheat by homologous cloning technology,and the expression pattern of TaHAK13 was analyzed by qRT-PCR technology.GUS staining was used to analyze the expression site of TaHAK13 in Arabidopsis thaliana.The function of TaHAK13 gene was verified by yeast and Arabidopsis thaliana.The interaction mechanism of TaHAK13 protein was analyzed by membrane system yeast two-hybrid and double luciferase complementary test.The main results are as follows:1.Bioinformatics analysis of TaHAK13RNA-seq data showed that TaHAK13-7DL had tissue expression specificity,which showed a high expression level in stems,leaves and grains,and a low expression in roots and spikes.The TaHAK13 gene in wheat has the closest evolutionary relationship with Hordeum vulgare HvHAK13 and Aegilops tauschii AetHAK13.Its encoded protein contains 11 transmembrane regions,Conservative domain analysis showed that TaHAK13 had the K_trans domain of HAK/KUP/KT family.Protein phosphorylation sites predict that TaHAK13 protein may be activated by upstream phosphorylase through serine/threonine residues,thus transmitting signals to downstream.Sequence analysis of promoter shows that the activity of TaHAK13 gene promoter is regulated by many factors.2.Expression analysis of TaHAK13 under abiotic stressThe expression of TaHAK13 gene in wheat roots under abiotic stress was analyzed by qRT-PCR.It was found that TaHAK13 responded to drought stress(20%PEG6000),low potassium stress(0.01 mM KCl)and high salt stress(200 mM NaCl).Under drought stress and low potassium stress,TaHAK13 was strongly induced to up-regulate its expression,and its expression level reached its peak at 3 h and 6 h respectively.Under salt stress,TaHAK13 was induced to up-regulate its expression,but its expression level was inferior to that of drought stress and low potassium stress.3.Analysis of tissue expression characteristics of TaHAK13The tissue expression characteristics of TaHAK13 were analyzed by GUS staining.At the seedling stage of Arabidopsis thaliana,TaHAK13 was mainly expressed in the veins,hypocotyls,main roots and lateral root tips of transgenic plants.At the mature stage of Arabidopsis thaliana,TaHAK13 is mainly expressed in veins,roots,stems and anthers,and the deeper the roots are tied,the deeper the dyeing is,which indicates that the expression of TaHAK13 in plants is affected by the development stage.4.Subcellular localization of wheat TaHAK13TaHAK13-GFP fusion expression vector was constructed to transform the transient expression of Agrobacterium tumefaciens in tobacco leaves.The subcellular localization of TaHAK13 was observed by laser confocal microscope,and it was found that TaHAK13 was located on the cell membrane and belonged to membrane protein.5.Functional analysis of TaHAK13 in yeast cellsTaHAK13 was transformed into yeast potassium sensitive mutant strain CY162.The results showed that TaHAK13 could restore the sensitive phenotype of CY162 on low potassium medium(1 mM KCl).Under the condition of low potassium stress,the yeast strain transformed with TaHAK13 and TaHAK1(control)has higher yeast growth rate and growth amount,intracellular K+content,external K+absorption rate and absorption amount than the yeast strain transformed with p416,which indicates that TaHAK13 and TaHAK1 have similar functions and have the function of transporting K+.When TaHAK13 was transferred into the salt-sensitive mutant strain AXT3K,it was found that only the positive control(TaSOS1)could grow normally under high salt conditions,but neither the strain transferred with TaHAK13 nor the empty vector(EV)could grow normally,which indicated that TaHAK13 had no Na+ transport function.6.Functional analysis of TaHAK13 in ArabidopsisTaHAK13 was transferred into Arabidopsis thaliana wild type WT and mutant athak5.The results showed that under the condition of c(K+)≥ 0.1mM,the root length,fresh weight and net K+flow rate of TaHAK13 transgenic line had no significant difference with the control line,but under the condition of c(K+)≤0.01 mM,the root length,fresh weight and net K+flow rate of TaHAK13 transgenic line were significantly higher than those of the control line.7.Analysis of TaHAK13 interaction mechanismThe membrane system yeast two-hybrid system was used to screen proteins interacting with TaHAK13.The self-activation and toxicity tests showed that the bait carrier TaHAK13-pBT3-N was nontoxic to the yeast system and had no self-activation activity.Five interacting proteins were identified after library screening,and their functions related to plant disease resistance,response to biotic and abiotic stresses,nutrient transport and so on.Two genes related to nitrogen transport(TaNRT5.10 and TaNRT6.3)were selected for peer-to-peer verification and double luciferase complementation test,and it was found that TaHAK13 interacted with TaNRT5.10 and TaNRT6.3.