Genome-wide Identification Of Trihelix Gene Family And Functional Study Of PgGT25-04 Gene In Panax Ginseng

Panax ginseng C.A.Meyer is a valuable Chinese herbal medicine of Araliaceae,and its main active ingredient is ginsenoside.Trihelix transcription factor family,also known as GT factor family,is a highly concerned gene family in plants,which plays an important role in plant growth and development,morphogenesis and biotic and abiotic stress response.At present,there are no reports about Trihelix transcription factor family in Panax ginseng.In this study,based on ginseng transcriptome database,bioinformatics methods were used to analyze the structure,chromosome location,gene replication,phylogeny,functional differentiation,expression patterns and network interaction of Trihelix transcription factor family members in Panax ginseng.At the same time,the changes of members of Trihelix transcription factor gene family and ginsenosides in hairy roots of Panax ginseng were studied by exogenous addition of methyl jasmonate(Me JA)and abscisic acid(ABA).Through the association analysis of "content and content","gene and content" and "gene and gene",we finally identified a negative correlation gene Pg GT25-04,which is closely related to the key enzyme genes in ginsenoside biosynthesis pathway.The interference vector p BI121-Pg GT25-04 containing Pg GT25-04 gene was successfully constructed.The ginseng explants were transformed by Agrobacterium tumefaciens,and the positive hairy root clones were successfully obtained.Ginsenosides were extracted by Soxhlet extraction,and the content of ginsenosides was detected by high performance liquid chromatography(HPLC).At the same time,the expression of Pg GT25-04 gene and key enzyme genes in ginsenoside biosynthesis pathway in positive hairy root clones were detected and analyzed by fluorescence quantitative PCR,and the function of Pg GT25-04 gene in regulating ginsenoside biosynthesis was preliminarily determined.The specific experimental results are as follows:1.Through screening and identification,a total of 218 Trihelix transcription factor family members were obtained and named PgGTs.2.Through evolutionary analysis,PgGTs are divided into five families,and members of the PgGTs family are evolutionarily conservative.According to the analysis of its structure,it is found that PgGTs have 20 motifs,and the genes in the same family have similar structure,but the structure is quite different among different families.When different family members are adjacent or close to each other in the same region on the chromosome or in the evolutionary tree,it is speculated that there is a tandem repetition relationship between family members,so they have similar or the same function.3.The GO functional annotation of PgGTs showed that there are 8 subfunctions,indicating that the function of Pg GT genes were widely distributed and had the binding function of transcription factors.4.Expression pattern analysis showed that the members of PgGTs gene family were spatio-temporal specific in 42 farm varieties,14 tissue sites and four different years of ginseng roots.Based on the analysis of PgGTs in the interaction network,it is found that different genes are clustered together to form a co-expression network,indicating that PgGTs perform gene function in a cooperative manner.5.After abscisic acid(ABA)treatment,six key enzyme genes in PgGTs and ginsenoside biosynthesis were selected for fluorescence quantitative analysis.It was found that PgGTs could respond to ABA treatment and had an effect on ginsenoside biosynthesis.It was preliminarily confirmed that PgGTs could participate in the regulation of abiotic stress response.6.After methyl jasmonate(Me JA)treatment,2 PgGTs were randomly selected from 5 subfamilies for gene expression study,and 6 genes with the same ABA treatment as above were selected in Me JA treatment.Compared with the two treatments,it was found that the gene expression was extremely significant in both ABA and Me JA treatments at 48 h.It is proved that PgGTs can respond to the regulation of Me JA and promote the bioaccumulation of ginsenosides after treatment for a certain time.7.Through WGCNA method,PgGTs were divided into blue-green,blue and brown modules to analyze the correlation with ginsenoside content,and the relationship between each module and ginsenosides content were determined.Furthermore,the PgGTs candidate genes related to ginsenoside biosynthesis were screened.Then the interaction network analysis was carried out with the key enzyme genes in the ginsenoside pathway,and finally a very significantly related Pg GT gene(Pg GT25-04)was identified for follow-up function research.8.Based on the analysis of physical and chemical properties and functional prediction of Pg GT25-04 gene,it was determined that Pg GT25-04 was a transcription factor negatively related to ginsenoside synthesis,and p BI121-Pg GT25-04 interference expression vector was constructed.9.The recombinant vector was transformed into ginseng explants by Agrobacterium tumefaciens induction,and 7 positive hairy root clones were obtained.Through the detection of ginsenosides,it was found that the contents of monomer saponins Rg1,Rd,PPT and PPD increased.The negative regulation of Pg GT25-04 gene in ginsenoside synthesis was verified by RNAi.In this study,we systematically analyzed the PgGTs gene family at the whole genome level,cloned a transcription factor gene involved in ginsenoside biosynthesis,and preliminarily verified that Pg GT25-04 gene can regulate ginsenoside biosynthesis.This lays a theoretical foundation for the study of functional genomics of Panax ginseng,provides a theoretical basis for further improving the synthetic pathway of ginsenosides,and provides rich genetic resources for the study of plant synthetic biology.