Study On The Function Of 14-3-3-like C Protein In Tobacco Plant

The 14-3-3 protein family is a highly conserved regulatory protein that is widely.present in eukaryotic cells and regulates ligand protein function by binding to phosphorylation sites.It is involved in a range of signal transductions,including control of metabolism,hormone signaling,cell differentiation,and biotic and abiotic stress responses.More and more studies have shown that 14-3-3 protein has an immune response function in plant-virus interactions.In the previous study,the laboratory screened a 14-3-3-like C protein with significant differences from different tobacco varieties infected with TMV.In order to explore its biological function in the process of stress resistance,the following research was conducted.(1)Bioinformatics analysis and subcellular localization of 14-3-3-like C protein The cloned 14-3-3-like C gene was isolated from tobacco leaves and analyzed by bioinformatics,The results showed that the full-length cDNA of the protein was 1027 bp,encoding 260 amino acids.The molecular weight of the protein was 58.26 kDa.The secondary structure consisted mainly of a-helix and random coil,which was characteristic of typical 14-3-3 protein.Its alignment with the 14-3-3 protein sequence in tobacco showed that the gene was closest to the 14-3-3-C1 type protein,and subcellular localization showed that the protein was distributed in both the tobacco cell membrane and the nucleus.(2)Screening the interaction proteins by yeast two-hybridWe screened two proteins that interact with 14-3-3-like C using yeast two-hybrid technique.The sequencing and alignment showed that the two proteins were 60S ribosomal protein L22-like and transcriptional mutant X2 protein.(3)Construction the transgenic tobacco plants of RNAi silencing 14-3-3-like CThe 14-3-3-like C protein gene expression vector pRNAi-14-3-3 was constructed and infected with tobacco by Agrobacterium.The F0 transgenic tobacco plants were obtained.(4)qPCR detects the effects on the photosynthetic system and resistance-related genes when the 14-3-3-like C gene is inhibitedqPCR detected two proteins interacting with 14-3-3,and the results showed that the expression levels of 60S ribosomal L22-like and transcriptional mutant X2 genes were significantly down-regulated.Detection of TMV resistance gene N and protein pathogenesis-related PR1-a gene expression levels in transgenic plants were down-regulated;Targeting molecules against TMV in pre-laboratory experiments are photosynthesis-related genes PetD?PsbA?PsaA?short-chain dehydrogenase TIC 32?Geranylgeranyl diphosphate reductase?oxygen-evolving enhancer protein 1,The detection results of these gene expression levels are shown that the genes PetD,PsbA and PsaA were significantly down-regulated in the transgenic plants.Short-chain dehydrogenase TIC 32,Geranylgeranyl diphosphate reductase,oxygen-evolving enhancer protein 1 expression down.It is suggested that the level of 14-3-3-like C protein gene expression affects photosynthesis,and it is speculated that this protein is associated with TMV infection tolerance.