Identification Of Panax Ginseng WRKY Gene Family And Study On Its Regulation Of Ginsenoside Biosynthesis

Panax ginseng,known as the most renowned medicinal plant globally,contains ginsenoside as its primary active ingredient.The biosynthesis of ginsenoside has been a popular research topic,with the molecular mechanism being of particular interest.Researchers have focused on the WRKY transcription factors,which are the largest class of plant-specific transcription factors.It plays a regulatory role in plant biological processes,such as responding to biotic or abiotic stresses,regulating plant growth and development,and participating in the synthesis of secondary metabolites.At present,there is no reported genome-wide analysis of the WRKY transcription factors in P.ginseng,and further research is needed to understand the mechanism of WRKY transcription factors in regulating the biosynthesis of triterpenoids in P.ginseng.As a result,it is crucial to examine how WRKY transcription factors transcribe and regulate ginsenoside synthesis.The main research contents and results are as follows:(1)In the P.ginseng genome,150 genes belonging to the WRKY family were identified.All 150 Pg WRKY genes were categorized into three primary groups and five subgroups in group II(IIa-IIe).Analysis of the gene structure and conserved motifs showed that the similarity in the distribution and characteristics of the Pg WRKY structural domains in the same group was extremely high.The analysis of cis-acting elements revealed that the majority of Pg WRKY gene promoters contain numerous cis-regulatory elements related to hormones and stress.To further investigate,we analyzed the expression patterns of Pg WRKY genes in 14 different tissues and their responses to high temperature,low temperature,salt,and drought stresses,utilizing publicly available databases.The expression type of Pg WRKYs could be divided into three groups: low-level expression,tissue-distinct and constitutive.The expression trends of genes changed significantly under high temperature,drought and low temperature treatment but slightly in response to salt treatment.(2)Transcriptome and metabolome sequencing were performed on the adventitious root(AR),fibrous root(GR)and callus(CT)of P.ginseng.Transcriptome sequencing yielded 7 Gb of data per sample.Compared with the AR and GR,a total of 15409 genes were differentially expressed.In the GR and CT groups,18475 genes were differentially expressed,while in the AR and CT groups,12936 genes were differentially expressed.Additionally,we detected a total of64 metabolites in the metabolome.Analyze the expression patterns of the Pg WRKY gene in different types of tissues,the correlation between the Pg WRKY gene and key enzyme genes for ginsenoside biosynthesis,and the correlation between the Pg WRKY gene and ginsenoside.Finally,13 Pg WRKY genes related to ginsenoside biosynthesis were screened.(3)Three Pg WRKY genes were cloned,including Pg WRKY12(g15107),Pg WRKY22(g22783),and Pg WRKY33(g28382).The c DNA sequences of these genes are 1374 bp,1029 bp,and 1551 bp in length,respectively.To determine the subcellular localization of these proteins,we constructed subcellular localization vectors PHB-Pg WRKY12,PHB-Pg WRKY22,and PHB-Pg WRKY33,and performed transient transformation in tobacco leaves.Our results indicate that all three proteins are located in the nucleus.(4)The yeast one-hybrid assays produced results indicating that Pg WRKY22 and 33 are capable of binding to the W-box promoter.The correlation analysis between Pg WRKY and ginsenoside biosynthesis pathway genes demonstrated a high correlation between the Pg WRKY gene and PPTS.Analysis revealed that the PPTS promoter sequence contains two W-box elements.The Dual luciferase reporter assays confirmed that Pg WRKY22 and 33 proteins are able to bind to the PPTS promoter and regulate its activity.