Systematic Analysis Of GRAS Gene Family In Jilin Ginseng And Functional Study Of PgGRAS68-01 Gene

The Jilin ginseng(Panax ginseng C.A.Meyer)is a perennial herb of the Araliaceae family,ginseng genus.It's well-known both at the home and abroad.Ginsenoside biosynthesis is controlled by structural genes and regulated by transcription factors.GIBBERELLIN-ACID INSENSITIVE,REPRESSOR of gai1-3 and SCARECROW(GRAS)transcription factor is a widespread transcription factor in a class of plants,which play an importantrole in the development of plant roots,meristem formation,gibberellin signaling,light signaling,biological and abiotic stress processes.GRAS transcription factor can be used as a tool to modify plant metabolic pathway.Through interaction with target gene promoter or regul atory element,it can regulate the expression of enzymegene,so as to activate multiple gene synergy in metabolic pathway and effectively improve the content of secondary meta bolites.However,there is no report on the related research of GRAS gene family on the identification,systematic analysis and involved in ginsenoside biosynthesis.In this study,based on the transcriptome database and expression quantity database of Jilin ginsengesta blished in the laboratory.the GRAS gene family in Jilin ginseng was identified,functional prediction,phylogenetic relationship,expression mode and network mutual system analysis.On this basis,the PgGRAS68-01 gene,which is closely related to ginseng saponin biosyn thesis,was screened out.Overexpression vector pBI121-Pg GRAS68-01 was constructed,and the aseptic seedlings of Panax ginseng were transformed by agrobacterium-mediated method.The positive hairy root strains of Panax ginseng were successfully obtained.The results are as follows:4.A total of 131 PgGRASs were identified to form the Jilin Ginseng GRAS(Pg GRASs)gene family data.Of these,59 PgGRASs have complete ORF.2.GO function annotation note display(Level 2),the main functions are concentrated in 15 categories: including cellular process(Cellular process),signaling(Signaling)and metabolic process(Metabolic process),multicellular organismal process(Multicellular organismal process),response to stimulus(Response to stimulus),regulation of biologi-cal process(Regul ation of biological process),developmental process(Developmental process),single-organism process(Single-organism process),binding(Binding),transcription factor activity(Transcrip tion factor activity),protein binding(Protein binding),nuclear acid binding transcription factor activity(Nucleic acid binding transcription factor activity),cell(Cell),organelle(Organelle)and cell part(Cell part).2.The results of ML(maximum-likelihood)evolutionary tree showed that the GRAS gene family of Panax ginseng was divided into 13 subfamilies in phylogenetic relationship,and the genes with similar conserved domain were clustered into the same subfamily,which indicated the diversity and conserved type of the evolution of the GRAS gene family of Panax ginseng at the same time.3.Evolutionary analysis shows PgGRASs divided into 13 sub-families.The conservative domain of the same subfamily gene has a high similarity.Explain that the evolution of the PgGRASs gene family is both diverse and conservative.4.By expression patterns analysis of PgGRAS genes of Jilin ginseng in 14 tissue,4 main roots of different years and 42 agricultural varieties,we found that PgGRASs has Spacetime specificity.Through interaction analysis,it was found that the PgGRAS genes formed a co-expression network.Substantiate that there is a collaborative relationship between them.5.Exogenous addition of gibberellicaic(GA)inhibits the growth and development of gins eng while reducing the content of saponinin in ginseng.It is speculated that the gibberelli caic pathway has inhibition effect on the synthetic pathway of ginseng saponin.Filtered to four genes associated with gibberellicaic response(Pg GRAS-44-04,Pg GRAS48-01,Pg GRA S50-01,Pg GRAS68-01).The expression patterns of these genes in ginseng hair roots under gibberellicaic processing were studied by qRT-PCR.Its function in the transduction path way of ginseng gibberellicaic signal is explored.6.Correlation analysis of PgGRASs expression with ginseng saponin synthesis key enzyme genes and different monomer saponin expression.Sifed out 10 genes.Then by analyzing the SNP mutation site,it was found that 2(Pg GRAS48-01 and Pg GRAS68-01)of these10 genes had significant changes.Among them.The Pg GRAS68-01 has a close inter-relatio nship with key enzyme genes.Finally,Pg GRAS68-1 is selected for further functional verif ication7.The full-length sequence(1656bp)of Pg GRAS68-01 gene was successfully cloned,and the overexpression vector pBI121-Pg GRAS68-1 was constructed.Conversion of ginseng seedlings using agrobacterium-mediated method mediated transformation,and obtain positive hairy roots.The Pg GRAS68-01 gene was initially demonstrated to be integrated into the ginseng genome using the PCR method.8.Test the saponin content of the single root system of the positively verified positive hairy roots.The suppressive effect function of Pg GRAS68-01 in ginseng saponin synthesis is verified.