Cloning and functional analysis of caffeic acid and rosmarinic acid glycosyltransferases from Arnebia euchroma / 中国中药杂志

Caffeic acid and its oligomers are the main water-soluble active constituents of the traditional Chinese medicine(TCM) Arnebiae Radix. These compounds possess multiple biological activities such as antimicrobial, antioxidant, cardiovascular protective, liver protective, anti-liver fibrosis, antiviral and anticancer activities. The phenylpropanoid pathway in plants is responsible for the biosynthesis of caffeic acid and its oligomers. Glycosylation can change phenylpropanoid solubility, stability and toxic potential, as well as influencing compartmentalization and biological activity. In view of the important role played by de-glycosylation in the regulation of phenylpropanoid homeostasis, the biosynthesis of caffeic acid and its oligomers are supposed to be under the control of relative UDP-glycosyltransferases(UGTs). Through the data mining of Arnebia euchroma transcriptome, we cloned 15 full-length putative UGT genes. After recombinant expression using the prokaryotic system, the crude enzyme solution of the putative UGTs was examined for the glycosylation activities towards caffeic acid and rosmarinic acid in vitro. AeUGT_01, AeUGT_02, AeUGT_03, AeUGT_04 and AeUGT_10 were able to glycosylate caffeic acid and/or rosmarinic acid resulting in different mono-and/or di-glycosylated products in the UPLC-MS analyses. The characterized UGTs were distantly related to each other and divided into different clades of the phylogenetic tree. Based on the observation that each characterized UGT exhibited substrate or catalytic similarity with the members in their own clade, we supposed the glycosylation abilities towards caffeic acid and/or rosmarinic acid were evolved independently in different clades. The identification of caffeic acid and rosmarinic acid UGTs from A. euchroma could lead to deeper understanding of the caffeic acid oligomers biosynthesis and its regulation. Furthermore, these UGTs might be used for regiospecific glycosylation of caffeic acid and rosmarinic acid to produce bioactive compounds for potential therapeutic applications.

Cloning and functional verification of carboxyl CoA ligases(AeCCLs) in Arnebia euchroma / 中国中药杂志

Carboxyl CoA ligases(CCLs) is an important branch of adenylate synthetase gene family, which mainly has two-step catalytic reactions. Firstly, in the presence of adenosine triphosphate, it can catalyze the pyrophosphorylation of carboxylateswith diffe-rent structures to form corresponding acyl adenosine monophosphate intermediates. Secondly, adenosine monophosphate was replaced by free electrons in the mercaptan group of enzyme A or other acyl receptors by nucleophilic attack to form thioesters. In this study, on the basis of the transcriptome database of Arnebia euchroma, two genes were selected, named AeCCL5(XP_019237476.1) and AeCCL7(XP_019237476.1). Bioinformatics analysis showed that their relative molecular weights were 60.569 kDa and 60.928 kDa, theoretical PI were 8.59 and 8.92, respectively. They both have transmembrane domains but without signal peptide. By multiple sequence alignment and phylogenetic tree analysis, we found that the similarity between AeCCLs and other plant homologous proteins was not high, and the substrate binding sites of AeCCLs were not highly conserved. The reasons might be that the sequence and structure need to adapt to the changes of new substrates in the process of evolution. In this study, the full-length of AeCCL5 and AecCCL7 were cloned into the expression vector pCDFDuet-1. The proteins of AeCCL5 and AeCCL7 with His-tag were expressed in Escherichia coli. The proteins of AeCCL5 and AeCCL7 were purified by nickel column. In vitro enzymatic reactions proved that both AeCCL5 and AeCCL7 can participate in the upstream phenylpropane pathway of shikonin biosynthesisby catalyzing 4-coumaric acid to produce 4-coumarin-CoA, and then to synthesis p-hydroxybenzoic acid, which is an important precursor of shikonin biosynthesis in A. euchroma.

Establishment of RNA interfered hairy root system of two CYP450 genes in Arnebia euchroma and its influence / 中国中药杂志

In this study, based on the transcriptome database of suspension cells of Arnebia euchroma, we explored two candidate cytochrome P450 enzyme genes that might relate to the shikonin biosynthesis downstream pathway when CYP76B74 sequence was referenced. We constructed interference-type hairy roots of candidate genes and cultured them. We measured the fresh weight, dry weight, total naphthoquinone content, shikonin and its derivatives content and expression levels of key enzyme genes involved in shikonin biosynthesis pathway. The effects of candidate genes on the growth and shikonin production of A. euchroma hairy roots were discussed, and the possible regulatory mechanisms that candidate genes affected shikonin synthesis were discussed. Through local Blast and phylogenetic analysis, two candidate CYP450 genes(CYP76B75 and CYP76B100) with high homology to CYP76B74 in A. euchroma were screened, and corresponding interference hairy roots were constructed. Compared with the control(RNAi-control), the fresh weight of CYP76B75 interfered hairy root(RNAi-CYP76B75) and CYP76B100 interfered hairy root(RNAi-CYP76B100) were significantly reduced, while dry weight were not affected, so the dry rate increased significantly. Except for β-acetoxyisovalerylalkannin, which is high in three groups of hairy roots, the contents of shikonin, deoxyshikonin, acetylshikonin, β,β'-dimethacrylicalkannin, β-hydroxyisovalerylshikonin,β-hydroxyisovalerylshikonin, isobutyrylshikonin and total naphthoquinones showed a consistent pattern: RNAi-CYP76B75>RNAi-CYP76B100>RNAi-control. Among them, the synthesis of β-hydroxyisovalerylshikonin was most significantly promoted by interfering with the expression of CYP76B75. The content of β-hydroxyisovalerylshikonin in RNAi-CYP76B75 was 11.7 times that of RNAi-control. RESULTS:: of real-time qPCR analysis showed that compared to RNAi-control, the expression levels of AePGT gene in RNAi-CYP76B75 and RNAi-CYP76B100 were not changed significantly, and the expression levels of CYP76B74 and AeHMGR were up-regulated. In addition, the expression level of CYP76B100 in RNAi-CYP76B75 was down-regulated, whereas in RNAi-CYP76B100, the expression of CYP76B75 was significantly up-regulated. Therefore, this study confirmed that when the expression of CYP76B75 and CYP76B100 were interrupted, the growth of hairy roots were suppressed, but the synthesis of shikonin were promoted. They might increase the shikonin biosynthesis by up-regulating the expression of CYP76B74 in the hairy roots of A. euchroma.

Core position of secondary metabolism of medicinal plants in ecological planting of Chinese materia medica and its utilization / 中国中药杂志

This paper summarized the effects of ecological planting on secondary metabolism firstly and pointed out that ecological planting can increase the content of secondary metabolites in plants, especially the content of defensive secondary metabolites. The possible mechanism was analyzed subsequently. Then, we reviewed the induction and utilization of secondary metabolism in the ecological planting of Chinese materia medica from the perspectives of biological control of pests and diseases, promotion of beneficial microorganism accumulation, optimization of mixed planting, regulation of no-tillage and straw cover. In this article, we pointed out that paying close attention to secondary metabolism is the most important feature of ecological planting of Chinese materia medica. Ecological planting can promote the accumulation of secondary metabolites of Chinese materia medica which means can improve the quality of Chinese materia medica, beneficial to the prevention and control of diseases, insects and weeds. Furthermore, lacking of systemic researches,the extensive verifications and systematic in-depth researches on the ecological planting of Chinese materia medica should be carry out urgently.