Transcriptome Analysis Of Wheat In Response To Potassium Stress And Functional Identification Of The Candidate Gene TaHAK25

Potassium participates in plant growth and development and physiological and biochemical processes in ion form,and plays an important role in plant response to abiotic stress.For a long time,plants have evolved a complex regulation mechanism of potassium uptake and transport to cope with the external environment of potassium deficiency.The response of plants to low potassium stress is mainly at the transcriptional level and post-translational regulation.In this study,transcriptome sequencing technique was used to understand the differences in transcriptional levels of wheat under different potassium stress,to find key functional genes,and to study the gene function and regulatory network under low potassium stress.The main results are as follows:(1)RNA-seq technique was used to study the differential expression of wheat variety Yunong 804 at transcriptional level under the treatments of total potassium(2 m M KCl,TK),low potassium(0.01 m M KCl,LK)and Potassium deficiency(0.01 m M KCl,DK).The parameters were set as threshold | log2(Fold Change)| > 1,padj < 0.05 to screen the differential genes.There were 4795 differentially expressed genes in LK vs TK comparison group,of which 4237 genes were up-regulated and 558 down-regulated.There were 2069 differentially expressed genes in NK vs TK comparison group,of which 1556 were up-regulated and 1013 were down-regulated.In the two comparison groups,there were 230up-regulated and 81 down-regulated common differentially expressed genes.Transcription factors and hormone-related genes were up-regulated in LK vs TK group,while Ros scavenging genes and photosynthesis-related genes were up-regulated in NKvs TK group,and the number of genes increased.Cluster analysis showed that differential genes were up-regulated in clusters Ⅰ,Ⅴ and Ⅵ under LK stress,and up-regulated in Ⅲ,Ⅳ and VI clusters under NK stress.Wheat had different expression patterns in low potassium and no potassium treatments,and there were differences in genes and regulatory mechanisms involved.(2)According to the analysis of RNA-seq according to FPKM > 100,there were 37 potassium transporter genes in LK treatment and 36 potassium transporter genes in NK treatment.Among the unknown expressed genes,Traes CS6A02G291900(TaHAK25,potassiumion transporter)was the most differentially expressed in the two comparison groups,and log2 Fold Change was 0.5958 and 0.75062 in the two comparison groups,respectively.The analysis of the structure and physicochemical properties of TaHAK25 gene showed that the gene contained 9 exons and 8 introns and encoded a basic protein consisting of 769 amino acids and 12 transmembrane domains.Homologous analysis showed that TaHAK25 gene was closely related to barley Hv HAK25 and rice Os HAK25 gene.q RT-PCR analysis showed that both low potassium and high salt stress could up-regulate the expression of TaHAK25 and the expression pattern was basically the same.Among them,the expression was up-regulated by about 8.6 times under low potassium stress and 3.2 times under high salt stress.Drought stress upregulated the expression of TaHAK25 gene within24 hours.The results showed that the expression of TaHAK25 gene was affected by low potassium stress and regulated by drought and high salt stress,so it belongs to stress response gene.(3)The functional complementarity test of yeast showed that the yeast strain CY162 with K~+ absorption deficiency transformed with TaHAK25 gene could grow normally on AP medium with 1 m M K~+ concentration,while the yeast transformed with empty vector could not grow.It is suggested that TaHAK25 can make up for the growth defect of CY162 in K~+uptake deficient yeast under the condition of low potassium concentration(1m M K~+),and has the ability to transport potassium ions.Subcellular localization showed that TaHAK25 protein was located on the cell membrane and belonged to membrane protein.(4)The main root development of TaHAK25 transgenic Arabidopsis plants was better in the low potassium stress medium of 0m M K~+ and 0.01 m M K~+,and the main root development of wild type was inhibited.The root length and fresh weight of transgenic Arabidopsis thaliana(athak5)lines were significantly different from those of wild type under low potassium stress medium of 0m M K~+,0.01 m M K~+ and 0.1m M K~+.In 0.01 m M K~+ medium,the content of MDA in transgenic Arabidopsis thaliana decreased,the content of proline increased significantly,and the damage of cell plasma membrane was less than that of the control.It is suggested that TaHAK25 gene can improve the tolerance to low potassium in Arabidopsis thaliana.(5)Four proteins with potential interaction with TaHAK25 protein were identified by wheat c DNA library screening.Luciferase complementary test was used to further verify that TaHAK25 protein could interact with Ta ACT(ACT domain-containing protein DS12,Traes CS7A02G294200)and Ta CRT(Calnexin homolog,Traes CS6B02G129800)proteins.Yeast one hybrid test and double luciferase activity test showed that Ta NAC71(NAC transcription factor 71)and Ta NAC74(NAC transcription factor 74)could positively regulate gene expression by binding to the promoter of TaHAK25.