Tobacco Nt14-3-3 Gene Involved In Low Potassium And High Salt Stress

14-3-3 protein is a highly conserved regulator in multicellular eukaryotic tissue.It plays an important role in regulating plant growth and development,participating in signal transduction and stress response by interacting with different target proteins.Therefore,14-3-3protein has attracted wide attention,but little research has been done on 14-3-3 protein in tobacco,and its molecular regulation mechanism is still unclear.Therefore,through a series of bioinformatics analysis and biotechnology,we studied the molecular mechanism of Nt14-3-3gene response to low potassium and high salt stress in tobacco,and drew six main conclusions.:（1）Two CDS sequences of 14-3-3 genes,783 bp and 759 bp in length,were amplified from the cDNA of common tobacco K326 by using 14-3-3 gene sequences of homologous species,and two full-length genomic sequences of 14-3-3 genes,5111 bp and 3069 bp in length,were obtained from the total DNA of K326.Bioinformatics analysis revealed that both14-3-3 proteins were acidic hydrophilic proteins without signal peptides and contained 9relatively conserved alpha helix structures,which may be related to the maintenance of protein structure and function.（2）qRT-PCR analysis results showed that two 14-3-3 genes were expressed in all tissues of tobacco rhizome,stem,leaf and flower,but the highest expression was in root.The results of expression analysis under stress showed that the expression levels of two 14-3-3 genes at different time points were significantly induced by abiotic stress factors.Only under ABA treatment,the expression patterns of the two genes were similar,while the response patterns of the two 14-3-3 genes to low potassium,high salt,H₂O₂ and 4℃stress were significantly different.The fusion expression vector pBWA（V）HS-14-3-3b-Glosgfp was constructed and injected into the leaves of native tobacco to observe the fluorescence.The results showed that the tobacco 14-3-3b protein was located in the cell membrane and nucleus.（3）14-3-3b in tobacco plant tolerance to low potassium studies showed that transgenic tobacco plants overexpressing 14-3-3b showed higher tolerance to low potassium stress.Specifically,under low potassium stress,the germination rate of transgenic tobacco seeds was significantly higher than that of wild type,and the main root and root hair grew longer.qRT-PCR was used to amplify the related marker genes involved in the response to low potassium stress.ABA biosynthetic gene,ROS scavenging gene and stress defense gene were significantly up-regulated in over-expressed plants,while potassium channel gene and potassium transporter gene were partially up-regulated.（4）Under the condition of high salt stress,the growth of over-expressed plants was better than that of wild-type plants at all growth stages.Compared with the wild type,chlorophyll content,proline content,K/Na content ratio were significantly higher,but H202accumulation was significantly less.（5）Seven proteins interacting with 14-3-3b were screened from 22 tobacco CIPKs by yeast two-hybrid test.A conservative binding motif that can interact with 14-3-3 protein was found in CIPKs interacting with 14-3-3b.The binding motifs contained in CIPK25 conform to mode I,i.e.（R/K）XX（pS/pT）XP;CIPK2,CIPK7,CIPK9,CIPK20,CIPK22,CIPK23conform to mode II,i.e.（R/K）XXX（pS/pT）XP.The interaction between 7 CIPK proteins and 5 potassium channel proteins was further detected.CIPK7,CIPK9 and CIPK25 interact with five potassium channel proteins,NKT2,NKT3,NKT4,NKT6 and NtKC1.（6）The Recombinant Prokaryotic expression vectors pEGX-4T-1-14-3-3b,pET28a-CIPK2,pET28a-CIPK7,pET28a-CIPK25 were successfully constructed and transformed into E.coli BL21（DE3）by double digestion.The target protein with a molecular weight of about 56 kDa was also detected by SDS-PAGE,which was consistent with the predicted molecular weight of 14-3-3b protein.Meanwhile,the target band with molecular weight of 54 kDa was detected,which was consistent with the predicted molecular weight of three CIPKs.The interaction between three CIPKs and 14-3-3b was further verified by pull-down test in vitro.（7）Transcriptome sequencing results showed that DEGs of transgenic and wild type under low potassium stress were mainly concentrated in carbon metabolism,amino acid biosynthesis,oxidative phosphorylation,RNA transport and regulation of transcription.Further screening of differentially expressed genes for qRT-PCR validation indicated that14-3-3b might play an important role in regulating the response of tobacco to low potassium stress by enhancing the transcription levels of CIPK protein kinase gene,potassium channel gene,potassium ion transporter gene and H⁺-ATPase gene.