Cloning and Functional Characterization of NADPH-Cytochrome P450 Reductases in Aconitum vilmorinianum.

Diterpenoid alkaloids (DAs) are major pharmacologically active ingredients of Aconitum vilmorinianum, an important medicinal plant. Cytochrome P450 monooxygenases (P450s) are involved in the DA biosynthetic pathway, and the electron transfer reaction of NADPH-cytochrome P450 reductase (CPR) with P450 is the rate-limiting step of the P450 redox reaction. Here, we identified and characterized two homologs of CPR from Aconitum vilmorinianum. The open reading frames of AvCPR1 and AvCPR2 were found to be 2103 and 2100 bp, encoding 700 and 699 amino acid residues, respectively. Phylogenetic analysis characterized both AvCPR1 and AvCPR2 as class II CPRs. Cytochrome c and ferricyanide could be reduced with the recombinant proteins of AvCPR1 and AvCPR2. Both AvCPR1 and AvCPR2 were expressed in the roots, stems, leaves, and flowers of A. vilmorinianum. The expression levels of AvCPR1 and AvCPR2 were significantly increased in response to methyl jasmonate (MeJA) treatment. The yeasts co-expressing AvCPR1/AvCPR2/SmCPR1 and CYP76AH1 all produced ferruginol, indicating that AvCPR1 and AvCPR2 can transfer electrons to CYP76AH1 in the same manner as SmCPR1. Docking analysis confirmed the experimentally deduced functional activities of AvCPR1 and AvCPR2 for FMN, FAD, and NADPH. The functional characterization of AvCPRs will be helpful in disclosing molecular mechanisms relating to the biosynthesis of diterpene alkaloids in A. vilmorinianum.

[Selection of optimal qRT-PCR reference genes for Aconitum vilmorinianum].

Screening suitable reference genes is the premise of quantitative Real-time PCR(qRT-PCR)for gene expression analysis. To provide stable reference genes for expression analysis of genes in Aconitum vilmorinianum, this study selected 19 candidate re-ference genes(ACT1, ACT2, ACT3, aTUB1, aTUB2, bTUB, 18S rRNA, UBQ, eIF2, eIF3, eIF4, eIF5, CYP, GAPDH1, GAPDH2, PP2A1, PP2A2, ACP, and EF1α) based on the transcriptome data of A. vilmorinianum. qRT-PCR was conducted to profile the expression of these genes in the root, stem, leaf, and flower of A. vilmorinianum. The Ct values showed that 18S rRNA with high expression level and GAPDH2 with large expression difference among organs were not suitable as the reference genes. NormFinder and geNorm showed similar results of the expression stability of the other candidate reference genes and demonstrated PP2A1, EF1α, and CYP as the highly stable ones. However, BestKeeper suggested EF1α, ACT3, and PP2A1 as the top stable genes. In view of the different results from different softwares, the geometric mean method was employed to analyze the expression stability of the candidate re-ference genes, the results of which indicated that PP2A1, EF1α, and ACT3 were the most stable. Based on the comprehensive analysis results of geNorm, NormFinder, BestKeeper, and geometric mean method, PP2A1 and EF1α presented the most stable expression in different organs of A. vilmorinianum. PP2A1 and EF1α were the superior reference genes for gene expression profiling in different organs of A. vilmorinianum.

De novo RNA sequencing and analysis reveal the putative genes involved in diterpenoid biosynthesis in Aconitum vilmorinianum roots.

In this study, the putative genes involved in diterpenoid alkaloids biosynthesis in A. vilmorinianum roots were revealed by transcriptome sequencing. 59.39 GB of clean bases and 119,660 unigenes were assembled, of which 69,978 unigenes (58.48%) were annotated. We identified 27 classes of genes (139 candidate genes) involved in the synthesis of diterpenoid alkaloids, including the mevalonate (MVA) pathway, the methylerythritol 4-phosphate (MEP) pathway, the farnesyl diphosphate regulatory pathway, and the diterpenoid scaffold synthetic pathway. 12 CYP450 genes were identified. We found that hydroxymethylglutaryl-CoA reductase was the key enzyme in MVA metabolism, which was regulated by miR6300. Transcription factors, such as bHLH, AP2/EREBP, and MYB, used to synthesize the diterpenes were analyzed. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02646-6.

Diterpenoid alkaloids from Aconitum vilmorinianum.

Diterpenoid alkaloids, named vilmorines A-D, in addition to fifteen known alkaloids, were isolated from roots of Aconitum vilmorinianum. Their structures were established on the basis of extensive spectroscopic analyses. Antibacterial and antioxidant studies on isolated compounds were also carried out.

Accelerated solvent extraction and pH-zone-refining counter-current chromatographic purification of yunaconitine and 8-deacetylyunaconitine from Aconitum vilmorinianum Kom.

This study aimed to seek an efficient method to extract and purify yunaconitine and 8-deacetylyunaconitine from Aconitum vilmorinianum Kom. by accelerated solvent extraction combined with pH-zone-refining counter-current chromatography. The major extraction parameters for accelerated solvent extraction were optimized by an orthogonal test design L9 (3)(4). Then a separation and purification method was established using pH-zone-refining counter-current chromatography with a two-phase solvent system composed of petroleum ether/ethyl acetate/methanol/water (5:5:2:8, v/v) with 10 mM triethylamine in the upper phase and 10 mM HCl in the lower phase. From 2 g crude extract, 224 mg of 8-deacetylyunaconitine (I) and 841 mg of yunaconitine (II) were obtained with a purity of over 98.0%. The chemical structures were identified by ESI-MS and (1)H and (13)C NMR spectroscopy.