Screening Proteins Interacting With StERF5 Transcription Factor Of Potato By Yeast Two-hybrid Technique

Transcription factors?TFs?are a DNA-binding protein that specifically binds to the cis-acting elements of the promoter region of the target gene to be modulated,thereby TFs regulat the expression degree,expression time,and tissue specificity of the target genes in the cell.Ethylene response factor?ERF?,as a subfamily of AP2/EREBP transcription factor family,is also a class of p lant-specific transcriptional regulatory factors.It is an important gene expression regulation protein in plant cell signal transduction systems.The abiotic stress response plays an important role in regulating gene expression in plants.Biological and abiotic stress play an important role in gene expression.Based on the preliminary work of the laboratory,this study cloned the gene of potato St ERF5 transcription factor and performed bioinformatics analysis.The yeast two-hybrid technology was used to screen the downstream interaction proteins of St ERF5 gene.For the first time,the functional interaction genes of potato StERF5 transcription factors were successfully excavated.The results obtained are as follows:1.The ERF5 gene sequence of potato transcription factor was obtained by electronic cloning,and specific primers with restriction enzyme sites were designed.The double-stranded c DNA was synthesized by RT-PCR using potato RN A of potato variety"zihuabai"as a template to amplify the transcription factor St ERF5 gene of potato.Sequencing results showed that the sequence was 100%homologous to the registered potato sequence in GenBank?XP₀15169356?.The sequence was 1240 bp in length,and the open reading frame was 738 bp.It belongs to the ERF class members of the EREBP subfamily and the encoded protein does not have a signal peptide and is a non-secretory hydrophilic protein.2.The St ERF5 gene fragment and the p GBK T7 vector were digested with Eco RI and Bam HI and ligated to construct the bait vector p GBKT7-St ERF5.The pGBK T7-StERF5 was introduced into the activated yeast strain Y2HGlod by the lithium acetate method.After one week,the colonies of the experimental group and the control group were tested for toxicity.The results showed that the number of colonies of the bait plasmid p GBKT7-StERF5 and the empty plasmid pGBKT7 on the SD/-Trp medium plate was almost the same,and they were measured at 5 h,10 h,15h,20 h,25 h and 30 h.The OD₆₀₀ were all greater than 0.8,indicating that the bait vector p GBKT7-StERF5 had no toxic effect on yeast Y2HGlod cells and could be used as a further yeast two-hybrid assay.3.The constructed bait vector pGBKT7-St ERF5 was transformed into yeast cells Y2HGlod for identification and analysis on different auxotrophic media.The results showed positive control pGBKT7-53+pGADT7 on SD/-Trp?tryptophan-defective?and SD/-Trp/X-?-Gal?tryptophan deficient supplemental X-?-Gal?solid media.PositivecontrolpGBKT7-53+pGADT7-Tandnegativecontrolsboth pGBK T7-Lam+p GADT7-T can grow normally.Therefore,it can be judged that the recombinant bait vector has no self-activation of the downstream reporter genes MEL1 and AUR1 of the yeast p GBK T7 vector.SD/-Trp/X-?-Gal positive control on the solid medium of Ab A?Aureobasidin?could grow outside,negative and no colony growth in the experimental group,indicating that the bait vector had no autoactivation effect on the MEL1 and AUR1 reporter genes.Thence,the bait vector pGBK T7-StERF5 constructed in this experiment can be used for the next screening interaction protein experiment.4.The bait vector pGBKT7-St ERF5 was co-transformed with the yeast library cDNA under drought stress in potato,which screened candidate protein interactions with the target protein,and finally screened and tested for three proteins interacting with potato St ERF5 in the NCBI database.In the above,cDN A sequencing was performed to confirm that the three proteins were histone H2B,specific protein phosphatase,and R3H type single-stranded nucleic acid binding protein.The analysis of the screened interaction proteins showed that they were highly homologous to tomato,which was as high as 90%.After GO analysis,three interacting candidate proteins were located in different cells and responded to the potato.Drought signaling metabolic pathways may have a role.5.By positively cloning the plasmid?pGADT7-X?and the constructed pGBK T7-StERF5 recombinant plasmid for rotational hybridization,once again identified that the histone H2B and the ERF transcription factor in the potato can indeed interact with each other,and further interaction mechanisms can be used in the future.Two-molecule fluorescence complementation and co-immunoprecipitation together validated how the candidate molecules interacted with each other to recruit their transcription factors for gene expression regulation.