Systematic Analysis And Functional Study Of Ginseng C2H2 Type Zinc Finger Protein Family

Ginsenoside is the main active ingredient of ginseng,which plays important roles in the treatment of cardiovascular diseases,cancer,and anti-oxidation.Ginsenosides are mainly biosynthesized by the mevalonate pathway(MVA)and methylerythrose phosphate(MEP)pathway.Many of the key enzyme genes involved in these biological processes have been studied,but the regulatory functions and mechanisms of transcription factors is uncertain.As one of the largest transcription factor families,C2H2 zinc finger proteins in plants function in plant growth and development,responding to biotic and abiotic stresses,and regulating secondary metabolism.In this study,based on the Jilin ginseng transcriptome database,the Jilin ginseng C2H2 zinc finger protein family was identified and analysed systematically,through these analyses,Pg ZFP59-03 was screened for regulating the biosynthesis of ginsenosides and its functions were verified.In this study,the systematic analysis and functional research of the Jilin ginseng C2H2 zinc finger protein family has filled the gaps in related research.The main findings are as follows:1.A total of 228 C2H2 zinc finger protein family transcripts alternatively spliced from under 115 genes were identified through bioinformatic analyses,and named Pg ZFPs.The molecular weight,hydrophilicity and hydrophobicity,instability index,fat index and other indicators of family members were investigated.2.Phylogenetic analysis and conservative motif analysis showed that the ginseng C2H2 zinc finger protein were divided into 6 subfamilies.Each subfamily has similar structures and is speculated to have similar functions.Through conservative motif analysis,we found that some of these play important roles in the biological processes.3.The GO function annotation of Pg ZFPs revealed that Pg ZFPs were annotated into 17 functional subcategories in three major functional areas,and were functionally significantly enriched in binding,transcription regulation,cell component,etc.Morever it is indicated that the C2H2 transcription factor family plays important roles in transcription regulation and so on.4.The expression pattern analysis of Pg ZFPs showed that the expression of family members in 4 different developmental stages,14 tissues and 42 genotypes are different,and it reflected the temporal and spatial specificity of family members in expression.5.We found that the family members constituted a huge co-experssion network,which reflects the synergistic expression of Pg ZFPs.6.The real-time quantitative PCR analysis showed that five Pg ZFPs response to salt stress,and through subsequent experiments,it was found that Pg ZFP59-03 may affect the synthesis of ginsenosides while responding to salt stress.7.Through bioinformatics analysis,Pg ZFP59-03 involved in ginsenoside biosynthesis was screened out,and the clonal hairy root lines overexpressing Pg ZFP59-03 and RNAi-Pg ZFP59-03 line were successfully obtained.8.The ginsenoside contents of hairy root lines overexpressing Pg ZFP59-03 were determined by HPLC,and it was found that Pg ZFP59-03 repressed the biosynthesis of ginsenosides.We quantified the expressions of some key enzyme genes and Pg ZFPs by q PCR,which showed that the expressions of key enzyme genes changed significantly through overexpressing of Pg ZFPs.It was further speculated that Pg ZFP59-03 may affect transcriptional regulation of key enzyme genes and perform biological functions.