Functional Identification Of Potato StWRKY57 Gene In Response To Drought Stress

The WRKY transcription factor is one of the largest families of transcription factors in higher plants,and it can regulate the target gene by interacting with the cis-acting elements of the target gene.WRKY transcription factor can regulate plant growth and development,biological metabolism and stress resistance process.Studies have found that overexpression of the WRKY gene can increase the tolerance of plants to drought,salinity,high temperatures and pathogens.However,there are few studies on signal transduction involved in potato WRKY transcription factor intervention.Therefore,this study constructed the overexpression and interference expression vector of potato WRKY gene and used yeast two-hybrid technology to screen for proteins interacting with the WRKY gene to study the biological characteristics of the WRKY gene and the mechanism of response to drought stress in potato,and provide a theoretical basis for further study of the metabolic pathway of WRKY gene in potato drought resistance.The main findings are as follows:1.Bioinformatics analysis of StWRKY57 in potato: StWRKY57 gene is located on chromosome 8,the CDS region length 762 bp,contains 4 exons.This gene encodes a total of 253 amino acids,which encodes a protein with a relative molecular weight of28.72 kDa,theoretical isoelectric point is 8.84,instability coefficient is 57.47,aliphatic index is 75.53,and the total average hydrophilicity is-0.653,indicating that the protein is a hydrophobic protein and the protein has a transmembrane structure.This protein is localized in the nucleus.The transient expression vector pEGFP-WRKY was successfully constructed by homologous recombination method,and the tobacco was transfected with agrobacterium-mediated transformation method,fluorescence was observed in the nucleus by laser confocal scanning microscopy.2.Construction of pCPB-WRKY57 overexpression vector and pCPB121-amiRWRKY57 interference expression vector by double enzyme digestion and plasmid recombination.Transgenic plants were obtained by genetic transformation,and the expression of StWRKY57 in transgenic plants was analyzed by qRT-PCR.Compared with non-transgenic plants,the relative expression of StWRKY57 gene wasup-regulated in overexpression transgenic plants,and the transformed lines of OE1-6were up-regulated 3.46,1.66,1.59,3.29,3.60 and 1.58 times,respectively.Compared with non-transgenic plants,the relative expression of StWRKY57 gene was down-regulated in artificial miRNA-interfering transgenic plants,and the transformed lines of RNAi1-6 were down-regulated 26%,69%,42%,67%,68% and 44%,respectively.3.The bait vector pGBKT7-WRKY was constructed by double digestion and plasmid recombination,and transformed into Y2 HGold yeast strain for toxicity and self-activation detection.It showed that the bait protein has no toxicity and self-activation ability,and can be screened for the next step.4.The protein interacting with pGBKT7-WRKY was screened by the Mating method,and a total of 14 positive hybrids were obtained.The 14 positive clones were found to be the same repeat gene,that gene is the glutamine synthetase gene.