Screening Of Low Potassium Tolerance Genes And Preliminary Analysis Of TaGRAS29 Transcription Factor In Wheat

K⁺ is an essential nutrient element for plant growth and development.When plants perceive the signal of low potassium stress,they can stimulate the expression of related transcription factors through signal transmission.The GRAS family is a plant-specific transcriptional regulator that plays an important role in plant growth and development and abiotic stress response.At present,there are few studies on the role of GRAS gene family in plant nutrition regulation.In order to investigate the role of GRAS gene in wheat under low potassium stress,a response gene TaGRAS29 of GRAS gene family was screened by analyzing the transcriptome data of low potassium stress in this study.The spatial structure,conserved domains,phosphorylation sites and phylogenetic relationships of TaGRAS29 were analyzed by bioinformatics.The TaGRAS29 promoter activity was analyzed by GUS staining.The expression pattern of TaGRAS29 was analyzed by qRT-PCR.The low potassium tolerance function of TaGRAS29 gene was verified by transgenic Arabidopsis Thaliana.Screening and validation of TaGRAS29 interacting proteins by yeast two-hybridization and isolation luciferase complementary assay.The main results obtained are as follows:1.In this study,transcriptomics was used to study differential genes in wheat seedlings treated with low K,and it was found that six types of transcription factors,MYb,WRKY,AP2,HLH,NAM and GRAS,were involved in wheat response to low potassium stress.TaGRAS29 was selected as the target gene in this study according to the GRAS gene family expression in the transcriptome.2.Bioinformatics analysis of TaGRAS29 showed that the ORF frame size of TaGRAS29 was 1713 bp,encoding 570 amino acids and containing more potential phosphorylation sites of serine and threonine.Tagras29 has the conserved domain of the GRAS gene family,and the amino acid sequence is highly variable in the N terminal.It has close evolutionary relationship with barley HvGRAS2H＿4 and rice OsCIGR1.Sequence analysis of promoter cis-acting elements indicated that the promoter activity of TaGRAS29 gene was regulated by many factors.3.Construct the GUS expression vector of the TaGRAS29 promoter,transform Arabidopsis Thaliana,and analyze the GUS activity of Arabidopsis Thaliana at the T₂ seedling stage.The results showed that the TaGRAS29 promoter could drive the expression of GUS reporter genes,and the TaGRAS29 promoter was active in the meristem regions of the leaf segments,leaves and root tips of S.erioides.4.qRT-PCR was used to study the function of TaGRAS29 under abiotic stress and its expression characteristics in different tissues of wheat.It was found that TaGRAS29 gene was responsive to low potassium stress（0.01 mM K⁺）,high salt stress（200 mM NaCl）and drought stress（20% PEG6000）.The transcription level of TaGRAS29 was up-regulated in both shoot and root of wheat（except that expression was not induced in upper ground tissues under drought stress）.TaGRAS29 was expressed in roots,stems,young leaves,flag leaves and seeds of wheat,with the highest expression in young leaves,followed by stem and seeds.5.The fusion expression vector of TaGRAS29 and GFP was constructed and transferred into tobacco leaves for transient expression.The laser confocal technique was used to study the subcellular localization of TaGRAS29,and it was found that TaGRAS29 was located in the nucleus and belonged to nuclear protein.6.When TaGRAS29 was transferred into wild type WT of Arabidopsis thaliana,root length and fresh weight of transgenic Arabidopsis thaliana were higher than those of wild type under low potassium condition,and there were significant differences,indicating that TaGRAS29 could enhance transgenic plants’ tolerance to low potassium stress.7.The protein interacting with TaGRAS29 was screened by yeast two-hybrid technique.The toxicity and self-activation activity of the decoy vector pGBKT7-TaGRAS29 showed that pGBKT7-TaGRAS29 had no toxicity and self-activation activity on the yeast system.Five potential interacting proteins related to plant disease resistance,nutrient factor response,biological and abiotic stress response were identified through library screening.Point-to-point interaction in yeast and isolated luciferase in plant showed that TaGRAS29 could interact with TaGF14 d and TaGLP.