Cloning And Functional Validation Of Squalene Epoxidase (PgSQE08-01) Gene In Panax Ginseng

Ginseng(Panax ginseng C.A.Meyer)is a perennial herb,which is a Chinese herbal medicine in Jilin Province.The main active ingredient of ginseng is ginsenoside.The biosynthesis of ginsenoside is not only regulated by transcription factors,but also controlled by structural genes.The molecular mechanism of ginsenoside biosynthesis has been a hot topic in the study of secondary metabolites of ginseng.Squalene epoxidase(SQE)is a key enzyme in the methanolic acid(MVA)pathway of plant secondary metabolites,which affects the biosynthesis of terpenoids secondary metabolites.However,it is not deep enough to study how SQE genes participate in the molecular mechanism of ginsenoside biosynthesis.This study was based on the established ginsenoside transcriptome database,expression database and ginsenoside content database.Bioinformatics was used to systematically analyze the key enzyme genes of ginsenoside SQE.Six PgSQE genes closely involved in ginsenoside biosynthesis were selected.Finally,PgSQE08-01 genes on ginsenoside biosynthesis was highly correlated.The full-length c DNA sequence of the gene was cloned by modern biotechnology,and we were successfully constructed the overexpression vector p CAMBIA3301-PgSQE08-01 and interference expression vector p ART27-PgSQE08-01 by the adventitious roots of ginseng as receptor material and Agrobacterium tumefaciens mediated genetic transformation was carried out.The positive root lines were obtained.The expression of related genes and the content of ginsenoside were determined by q RT-PCR and HPLC.The function of PgSQE08-01 genes involved in ginsenoside biosynthesis was studied.The results were as follows:1.20 PgSQE genes were obtained from Jilin ginseng transcriptome database,Chinese genome database and Korean genome database.2.GO functional annotation results showed that PgSQEs genes were annotated to8 functional subclasses at the level 2.Enrichment analysis showed that PgSQEs genes were significantly enriched in metabolic processes,binding and catalytic activities.3.PgSQEs gene and ginsenoside synthesis key enzyme gene interaction ne twork analysis showed that the PgSQEs with SS-1,Pg CYP137,Pg CYP311,Pg CYP339,DS-1,DS-3,AS-1,AS-6,CAS-21,CAS-22,CAS-23,FPS-22,and UGT71A27-2 genes are closely related,and the interaction is involved in the metab olic process of plants.The PgSQEs gene expression level and ginsenoside cont ent was analyzed.The results showed that the expression of 6 PgSQEs genes was significantly related to ginsenoside content,and 6 sequences had complete ORF and conserved domains.Under different P value conditions(1.00 E-08 ≤P ≤5.00 E-02),the PgSQE08-01 always had a close interaction with key enzy me genes,and finally determined the in-depth functional study.4.We obtained full-length sequences of PgSQE08-01 genes and the length of 1,626 bp.The hyperexpression vector p CAMBIA3301-PgSQE08-01 and inter ference vector p ART27-PgSQE08-01 were constructed successfully,twelveover-e xpressed hairlike root clones were obtained by genetic transformation using gin seng adventitious roots as acceptor material,among which four were positive h airlike root clones.Ten hair root clones with interference expression were obtain ed,of which two were positive.5.The content of hyperexpressed positive root ginsenoside was determined by HPLC.The results showed that the PgSQE08-01 gene increased the content of five monomer ginsenoside Rb1,Rb2,Rb3,Rd,Re,and total ginsenoside significantly.It was preliminarily determined that PgSQE08-01 could affect the biosynthesis of ginsenoside.6.The expression of PgSQE08-01 genes and Six key enzyme genes(FPS-22,CYP-311,SE2-1,UGT27-2,DS-3,and CAS-21)in the Over-expression of positive hairlike root clones was determined by fluorescence quantitative RT-PCR.The expression level of 5 key enzyme genes(FPS-22,CYP-311,SE2-1,UGT27-2,and DS-3)in ginsenoside biosynthesis could be significantly improved,which the PgSQE08-01 genes could be involved in ginsenoside biosynthesis.Hence,this study explored the function,evolution,and expression activitiesof SQE genes in ginseng,which not only provided a molecular theoretical basis of important functional genes research in ginseng,but also provided genetic resources for synthetic biology of ginsenoside biosynthesis.