Study On AP2/ERF Transcription Factors And Histone Deacetylase Synergistically Regulate The Synthesis Of Terpene Phenolic Compounds In Hemp

Hemp(Cannabis sativa.L)is a medicinal plant with a long history,and some terpene phenolic secondary metabolites enriched in hemp flowers and bracts have important medicinal value.Among them,CBD has the effect of treating epilepsy,analgesia and so on,CBD is the terpene phenolic compound with the highest medicinal value.However,Cannabidiol Cannabidiolic(CBD)and Cannabichromen(CBC)share the Cannabigerolic acid(CBGA)precursor,which is competitive in the metabolic flow relationship.How to improve the CBD in the biosynthetic pathway and reduce the content of other minor terpene phenolic compounds is the focus and difficulty of the research.The elucidation of the regulatory mechanism of secondary metabolite synthesis is the basis of synthetic biology research.Combining genomic and transcriptome to excavate key enzyme genes for terpene phenolic compounds synthesis,screening for transcription factors regulating key enzyme genes and resolving regulatory mechanisms are effective methods for synthetic regulation research.In previous studies,the key enzyme genes in the biosynthetic pathway of terpene phenolic compounds had been elucidated.In this paper,combining the genome of hemp and the transcriptome of different tissues and organs,we screened APETALA2/Ethylene-Responsive(AP2/ERF)transcription factors that may regulate the synthesis of terpene phenolic compounds and the transcriptional cofactor histone deacetylase(HDAC).and studied the mechanism of their synergistic regulation of terpene phenolic synthesis.The following results have been obtained in this study:1.Screening AP2/ERF transcription factors related to the synthesis of terpene phenolic compoundsCombined with weighted gene co-expression network analysis(WGCNA analysis),correlation analysis between AP2/ERF transcription factors and key enzyme genes,and the specific expression of AP2/ERF transcription factors in different organs,we screened 3 AP2/ERF transcription factors preliminarily(Cs DREB18,Cs ERF04,Cs AP2-14).They may be involved in the synthesis of terpene phenolic compounds.Binolecular fluorescein reporter experiments on the promoters of three candidate AP2 / ERF genes and key enzyme genes revealed that Cs AP2-14 interacts with Cs OAC,Cs DREB18 interacts with both Cs OAC and Cs CBGAS,and Cs AP2-14 and Cs DREB18 may be involved in the regulation of terpenoid phenolic compound synthesis.2.Identification of the HDAC gene family of hempIn this study,14 HDAC gene family members of hemp were identified.And we analyzed the physicochemical properties,gene structure,chromatin localization,conserved domains,alternative splicing,cis-acting elements,and phylogenetic relationships of all members.We master the basic information of HDAC and the functions and characteristics of this familial nature,laying a foundation for the mining of target genes and functional validation..3.Screening of HDACs related to the biosynthesis of terpene phenolic compoundsFirst,we analyzed the gene expression patterns of HDAC in different organs(verified by q RT-PCR),the correlation with key enzyme genes,and the HDAC homology reported to be involved in secondary metabolism regulation Further,we treated hemp with histone deacetylase inhibitors(trostatin A,Trichostatin A,TSA),and performed qRT-PCR analysis of all HDAC genes and terpene phenolic compound synthesis pathways,and proved that TSA could effectively inhibit the expression of HDAC genes and affect the expression of key enzyme genes.In conclusion,four HDAC(Cs HDA1,Cs HDA7,Cs HDA10,Cs HDT1)that may be related to the synthesis of terpene phenolic compounds were initially selected.We cloned the candidate HDAC genes and observed their subcellular localization,and found that both Cs HDA1 and Cs HDA10 were localized to the nucleus.4.Verify the interaction between AP2/ERF and HDACThrough luciferase complementation(BiFC)experiments of candidate AP2/ERF transcription factors and histone deacetylases in tobacco,it was found that CsDREB18 and CsHDA10 have an interacting relationship.So they may coordinately regulate the synthesis of terpene phenolic compounds.