Genome-wide Analysis And Functional Verification Of The Chloride Channel TaCLCs Family Gene In Wheat

Nitrogen is one of the three essential nutrient elements for plant growth and development.Low nitrogen content in soil is the main factor restricting crop growth,and also affects crop yield and quality.In order to explore the genes related to nitrogen absorption and utilization,this study obtained the TaCLC family genes of low nitrogen stress response chloride ion channel protein by transcriptome analysis technology.The TaCLC family genes were identified by wheat database,and analyzed by bioinformatics methods.The functions of some TaCLC genes were analyzed by yeast mutants and Arabidopsis thaliana,and the interaction proteins and upstream regulatory transcription factors of TaCLCs were identified.The main results are as follows :(1)Transcriptome technique was used to analyze the expression of differentially expressed genes in wheat seedlings under nitrogen starvation stress,and the expression patterns of differentially expressed genes were analyzed.GO and KEGG databases were used to annotate the differentially expressed genes.Wheat TaCLC family genes related to nitrogen uptake and utilization were found in transcriptome data,and the transcriptional expression profiles of TaCLC family genes in response to nitrogen starvation were obtained.(2)The wheat genome database was searched by using the HMM model of voltage-gated chloride channel(CLC),and the wheat genome database was searched by Blastp with the CLC gene of Arabidopsis thaliana as the reference sequence,a total of 23 TaCLC gene sequences were identified.Based on the phylogenetic analysis of the TaCLC gene family,it was found that the family was mainly divided into two subfamilies(Ⅰ and Ⅱ)and seven subfamilies(-a,-c1,-c2,-e,-f1,-f2,-g).The distribution of TaCLC family genes in wheat chromosomes A(8),B(7)and D(8)was relatively balanced,and most homologous genes had collinearity.There is no collinearity among some homologous genes,which indicates that the fragment loss and gene loss of homologous genes occur simultaneously during the evolution of the family genes.The analysis of gene structure,protein structure and conserved motifs showed that TaCLCs gene contained 3-8 introns,4-9 exons and 8-12 transmembrane regions.For the same cluster of homologous genes,the motif structure and number were basically similar,but the conserved motifs of-a,-c1,-c2,-g2 clusters and-e,-f1,-f2 clusters were different.(3)One gene was selected from each of the seven clusters analyzed by TaCLC gene family and the gene expression changes under Na Cl stress and low nitrogen stress were studied by q RT-PCR.Except TaCLC-c2 gene cluster,the gene family could respond to the up-regulation of low nitrogen stress and Na Cl stress.The expression of TaCLC-a-6AS-1 gene was the highest in wheat roots under low nitrogen stress for 6 h.The relative expression of TaCLC-c1-3AS,TaCLC-e-3AL and TaCLC-f2-2DL genes at 24 h after Na Cl stress were higher than other genes.(4)The TaCLCs-p1300-GFP vector was constructed and transformed into wheat protoplasts using PEG transformation solution.The expression positions of TaCLC-a-6AS-1,TaCLC-c1-3AS and TaCLC-e-3AL were observed under the confocal microscope.The proteins encoded by TaCLC-a-6AS-1 and TaCLC-c1-3AS were expressed on vacuole membrane,and the proteins encoded by TaCLC-e-3AL were expressed on chloroplast.(5)The yeast recombinant expression vectors TaCLC-a-6AS-1-p416,TaCLC-c1-3AS-p416 and TaCLC-e-3AL-p416 were transformed into yeast mutant strain YJR040 W,respectively.The mutant strain lacked the chloride ion channel gene,and its Cl-or NO3-transport ability was blocked,and growth was inhibited.The transgenic yeast strains could grow normally on YPD+1 M Na Cl,YPD+1 M KCl and YPD+1 M KNO3 solid medium,indicating that the proteins encoded by these three genes had Cl-and NO3-transport functions.(6)The plant expression recombinant plasmids TaCLC-a-6AS-1-p1300,TaCLC-c1-3AS-p1300 and TaCLC-e-3AL-p1300 were transformed into Arabidopsis thaliana,respectively.The overexpression lines and WT were cultured on MS medium with different NO3-concentrations.The root length and fresh weight of Arabidopsis thaliana overexpressing TaCLC-a-6AS-1 and TaCLC-c1-3AS were higher than those of WT on MS medium and low nitrogen MS medium.The NO3-accumulation of TaCLC-a-6AS-1,TaCLC-c1-3AS and TaCLC-e-3AL plants grown on MS medium was significantly higher than that of WT,indicating that these three genes have nitrogen absorption and transport functions.(7)The proteins TaPRK,TaVDAC and TaSn RK1α3-B interacting with TaCLC-a-6AS-1 and TaCLC-c1-3AS were screened by yeast two-hybrid technique.The interaction between proteins was verified by yeast point-to-point hybridization test and double luciferase complementation test.In yeast point-to-point verification,Y2 H yeast containing BD-Bait plasmid and Prey-AD plasmid could grow on SD/-Trp/-Leu,SD/-Trp/-Leu/-His and SD/-Trp/-Leu/-His/-Ade defect medium.In the dual luciferase complementation test,there was an obvious LUC signal in the injection area of the test group,while no fluorescence signal was detected in other negative control areas.It indicated that TaCLC-a-6AS-1 and TaCLC-c1-3AS proteins interacted with TaPRK,TaVDAC and TaSn RK1α3-B proteins.(8)The upstream regulatory transcription factors TaWRKY81 and TaWRKY96 of this gene were screened by yeast single hybridization.In the yeast point-to-point experiment,Y1 H yeast containing Bait-p Ab Ai plasmid and Prey-AD plasmid could grow on SD/-Leu+Ab A300 inhibitory defect medium.Dual luciferase reporter assay showed that TaWRKY81 and TaWRKY96 could up-regulate the promoter activity of TaCLC-a-6AS-1 by about 2 and 3.5 times,respectively.It indicated that transcription factors TaWRKY81 and TaWRKY96 had regulatory effects on TaCLC-a-6AS-1.