Function Research Of The AeHMGR Gene With The Key Enzyme Of Saponin Synthesis In Aralia Elata(Miq.) Seem

Aralia elata(Miq.)Seem is one of the most important medicinal plants of Araliaceae.Saponin is the main active substance,which has effects of anti-tumor,anti-diabetes,liver protection and heart protection.The Aralia elata metabolism of saponins mainly depends on mevalonic acid(MVA)pathway,of which 3-hydroxy-3-methylglutaryl-Co A reductase(HMGR)is the first rate-limiting enzyme in this pathway.In this study,the callus of A.elata treated with methyl jasmonate was transcriptome sequenced.Based on the transcriptome data,Ae HMGR,the first key enzyme gene of MVA pathway,was identified and screened out.By constructed plant overexpression vector,Ae HMGR gene was heterologous expression in tobacco and overexpression in A.elata.The expression of key enzyme genes in transgenic tobacco(Nt FPS,Nt SS,Nt SE)and key enzyme genes in overexpression A.elata(Ae FPS,Ae SS,Ae SE,Aeβ-AS)by q RT-PCR.HPLC was established to detect five saponins of Aralia elata.The content of saponins in body embryos of overexpressed A.elata was analyzed.To explore the function of Ae HMGR gene in the MVA pathway of saponin metabolism.The main results are as follows:(1)RNA-Seq analysis showed that there were 586 differentially expressed genes in methyl jasmonate treated callus and untreated control callus.KEGG analysis was mainly concentrated in plant hormone signal transduction and other metabolic pathways.GO analysis mainly enriched in the secondary metabolic process,secondary metabolite,jasmonic acid response,etc.Eleven key enzyme genes were screened in MVA pathway,which two were Ae HMGR genes.(2)The length of the Ae HMGR sequence is 1065 bp.The protein is a hydrophobic protein,which locates on the mitochondrial membrane and belongs to the HMGR superfamily.Ae HMGR gene is expressed in different tissues of A.elata,and is highly expressed in somatic embryos and stems,showing obvious tissue specificity.(3)The plant expression vector p ROKII –Ae HMGR was successfully constructed.The gene was transferred into tobacco by leaf disc transformation and 6 transgenic tobacco lines were regenerated.The gene expression of Nt SS,Nt FPS and Nt SE in transgenic tobacco was significantly lower than that of the wild type,and the lowest was about 0.24 times of that of the wild type.(4)The Ae HMGR gene was transferred into A.elata,and ten of overexpressed embryogenic callus lines and five of overexpressed somatic embryo lines were regenerated.The expression of Ae FPS,Ae SE,Ae SS and Aeβ-AS genes in overexpressed embryogenic callus was significantly lower than that of wild type,and the lowest was about 0.3 times of wild type.The expression of Ae HMGR gene in overexpressed somatic embryos was significantly higher than that in the wild type,and the expression of Ae FPS and Ae SS genes were decreased,while the expression of Ae SE and Aeβ-AS genes were increased.The expression of Ae SE and Aeβ-AS gene in somatic embryo line 7 was 11.51 and 9.38 times of that in wild type.(5)The HPLC method was established for five saponins in A.elata,and the content of saponins in the Ae HMGR overexpressed somatic embryos was determined by using this method.The content of total saponins in line 7 was 1.14 times of that in the wild somatic embryo and 0.30 times of that in wild type leaf,but the content of oleanolic acid in line 7 was1.35 times of that in wild type leaf.