Functional Verification And Regulatory Mechanism Of Potassium Transporter TaHAK1 And TaHAK5 Genes In Wheat

Potassium is one of the essential nutrients for wheat growth and development.Improving the utilization efficiency of potassium fertilizer is conducive to the high and stable yield of wheat.The KUP/HAK/KT family mediates the transmembrane transport of potassium ions in plants.In our previous study,the high-affinity potassium transporter genes TaHAK1b-2BL and TaHAK5-3DL were cloned from the potassium-efficient wheat variety Yunong 804.It was found that both TaHAK1 and TaHAK5 genes belonged to Cluster I in the KUP/HAK/KT family.In this study,the functions of TaHAK1 and TaHAK5 were further verified in Arabidopsis thaliana.The expression site of TaHAK5 in Arabidopsis thaliana seedlings was analyzed by GUS tissue staining.The regulation mechanism of TaHAK5 in plants was preliminarily explored by membrane system yeast two-hybrid and yeast one-hybrid experiments,which laid a foundation for the study of the molecular mechanism of wheat potassium uptake.The main results are as follows :1.Functional analysis of TaHAK1 and TaHAK5 in Arabidopsis thalianaTaHAK1 and TaHAK5 were transformed into Arabidopsis wild-type WT,single mutant athak5 and double mutant athak5/akt1 by floral dip method.The results showed that under the condition of sufficient K+(1 m M),the root length,malondialdehyde content and proline accumulation of TaHAK1 and TaHAK5 transgenic Arabidopsis lines were not significantly different from those of the control lines.Under the condition of insufficient K+ concentration(0.01 m M,0 m M),the root length and proline accumulation of TaHAK1 and TaHAK5 transgenic Arabidopsis lines were significantly higher than those of the control lines.The malondialdehyde content was significantly lower than that of the control lines.It indicated that TaHAK1 and TaHAK5 had the function of promoting plant root growth and absorbing and transporting K+ in low potassium environment,and reduced the content of MDA in plants after low potassium stress,increased the accumulation of proline,and enhanced the tolerance of plants to low potassium stress.2.Salt tolerance analysis of TaHAK1 and TaHAK5 in yeastTaHAK1 and TaHAK5 were transformed into potassium-sensitive mutant yeast strain CY162 and cultured on AP-U medium with normal potassium(10 m M K+)and low potassium(0.1 m M K+)with gradient concentration of Na+.The results showed that when the K+concentration was sufficient(10 m M),all the transformed yeasts grew well.With the increase of Na Cl concentration to 750 m M,the empty yeasts did not grow,while the TaHAK5 and TaHAK1 yeasts could grow.When the concentration of K+ was low(0.1 m M),the yeast transformed with empty vector could not grow,while the yeast transformed with TaHAK5 and TaHAK1 could grow normally,even on the medium with 500 m M Na Cl,and the yeast transformed with TaHAK5 grew better than TaHAK1.These results indicated that TaHAK5 is a high affinity K+ absorption protein insensitive to Na+.3.Screening of interaction proteins between TaHAK1 and TaHAK5TaHAK1 and TaHAK5 were transformed into NMY51 yeast strain respectively,and the proteins interacting with them were screened by membrane system yeast two-hybrid technology.The detection of toxicity and self-activation activity showed that the bait vectors TaHAK1-p BT3-N and TaHAK5-p BT3-N had no toxicity and self-activation activity to this system,and could be screened.Through library screening,21 proteins that may interact with TaHAK1 and 21 proteins that may interact with TaHAK5 were identified.The functions of these genes involve plant disease resistance,signal transduction,organ morphogenesis,stress resistance and other aspects.The protein TaNHL interacting with TaHAK1 and the protein TaGRF1-D interacting with TaHAK5 were verified by yeast point-to-point test and dual luciferase complementation test.4.Analysis of cis-acting elements and tissue expression characteristics of TaHAK1 and TaHAK5 promotersThe cis-acting elements contained in the 2000 bp sequence before ATG of TaHAK1 and TaHAK5 genes were analyzed using the online website Plant CARE.The results showed that there were 15 identical cis-acting elements in the promoter regions of these two genes,which were related to hormone response,light response and stress response.There were 5 specific motifs in the TaHAK1 promoter and 11 specific motifs in the TaHAK5 promoter.The promoter sequences of TaHAK1 and TaHAK5 genes were ligated to the GUS vector.The pre-test results of onion epidermal cells showed that blue was observed in the onion epidermal cells transformed with p TaHAK5-GUS,while the onion epidermal cells transformed with p TaHAK1-GUS did not turn blue,indicating that the promoter of TaHAK5 had promoter activity and the promoter activity of TaHAK1 was low in onion epidermal cells.The promoter TaHAK5 was transformed into Arabidopsis thaliana.The results showed that the GUS driven by the TaHAK5 promoter was mainly expressed in the stomata and hypocotyls of Arabidopsis seedlings,but not in the roots.5.Screening of upstream regulatory proteins of TaHAK5The TaHAK5 promoter fragment was transferred into Y1 HGold yeast,and the upstream regulatory transcription factors were screened by yeast one-hybrid technique.Two transcription factors TaWRKY96 and TaNAC71 were obtained by library screening.Yeast point-to-point verification test showed that Y1 H yeast containing prey vector plasmid and TaWRKY96-AD plasmid could grow on SD/-Leu + Ab A500 inhibition defect medium.The dual luciferase reporter gene activity assay showed that TaWRKY96 could increase the activity of TaHAK5 promoter by nearly 3 times.It shows that TaWRKY96 has a regulatory effect on TaHAK5.