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.
Boraginaceae/genetics* ; Cloning, Molecular ; Coenzyme A ; Coenzyme A Ligases/genetics* ; Ligases ; Phylogeny

This study aimed to explore the correlation between rhizosphere soil microorganisms of wild Arnebia euchroma and the content of medicinal components to provide guidance for the selection of the ecological planting base. The total DNA of rhizosphere soil microorganisms of wild A. euchroma was extracted, and the microbial community structure of rhizosphere soil microorganisms was analyzed by IlluminaMiseq high-throughput sequencing technology. The content of total hydroxynaphthoquinone pigment and β,β'-dimethylacrylalkannin in medicinal materials was determined by high-performance liquid chromatography(HPLC). The physicochemical pro-perties of rhizosphere soil of wild A. euchroma in main producing areas were determined, and the correlation of soil microbial abundance with index component content and soil physicochemical properties was analyzed by SPSS software. The results showed that the species composition of rhizosphere fungi and bacteria in A. euchroma from different habitats was similar at the phylum and genus levels, but their relative abundance, richness index(Chao1), and community diversity(Simpson) index were different. Correlation analysis showed that the content of available phosphorus in soil was positively correlated with the content of total hydroxynaphthoquinone pigment and β,β'-dimethylacrylalkannin, and the abundance of five fungal genera such as Solicoccozyma and six bacterial genera such as Pseudo-nocardia and Bradyrhizobium was positively correlated with the content of medicinal components in medicinal materials. The abundance of Bradyrhizobium was significantly positively correlated with the content of β,β'-dimethylacrylalkanin. The abundance of fungi such as Archaeorhizomyces was significantly positively correlated with the content of available phosphorus in rhizosphere soil, and Bradyrhizobium was significantly negatively correlated with soil pH. Therefore, the abundance of fungi and bacteria in the rhizosphere of A. euchroma has a certain correlation with the medicinal components and the physicochemical properties of the rhizosphere soil, which can provide a scientific basis for the selection of ecological planting bases in the later stage.
Rhizosphere ; Soil Microbiology ; Bacteria/genetics* ; Phosphorus ; Soil ; Boraginaceae

Arnebia euchroma is the main source for medicinal herb Zicao. and its most important component shikonin compounds have high medicinal and industrial value. This research is aimed to build overexpression vectors and RNAi vectors for key secondary metabolism genes of A. euchroma, and bulid platform for constructions of related transgenic lines using GATEWAY technology. To build genetic material based genetic research platform is to provide a great convenience for digging and functional verification of the genes on secondary metabolic pathway, and also to fill the gaps in transgenic research of A. euchroma. This study is also important for the cultivation of shikonin high-yielding strains of A. euchroma.
Boraginaceae ; genetics ; metabolism ; Genetic Vectors ; genetics ; metabolism ; Plant Proteins ; genetics ; metabolism ; RNA Interference ; Secondary Metabolism

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.
Boraginaceae/genetics* ; Caffeic Acids ; Chromatography, Liquid ; Cinnamates ; Cloning, Molecular ; Depsides ; Glycosyltransferases/genetics* ; Phylogeny ; Tandem Mass Spectrometry

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.
Boraginaceae ; genetics ; Cytochrome P-450 Enzyme System ; Naphthoquinones ; Phylogeny ; Plant Roots ; RNA ; RNA Interference

A cDNA sequence of Arnebia euchroma AP2/ERF named AeAP2/ERF was cloned by in silico cloning in this study, using ACX71873 sequence from Lithospermum erythrorhizon as the probe sequence. Some characters of the AP2/ERF gene and encoded protein sequences were predicted and analyzed by the bioinformatics methods, including general physical and chemical properties, hydrophobieity, signal peptide, secondary structure, localization sites in cells. Results showed that the 876 bp long gene included a 1 077 bp ORF and encoding 205 amino acid. The AeAP2/ERF protein had no signal peptide, it was a hydrophilic proteins located in nucleus. The function of the AP2/ERF protein was mainly involved with metabolism controlling and signal transduction.
Amino Acid Sequence ; Base Sequence ; Boraginaceae ; classification ; genetics ; Cloning, Molecular ; Computational Biology ; Computer Simulation ; Hydrophobic and Hydrophilic Interactions ; Molecular Sequence Data ; Phylogeny ; Plant Proteins ; genetics ; Protein Structure, Secondary ; Protein Structure, Tertiary ; Transcription Factors ; chemistry ; genetics

Twenty-seven ERF transcription factor family genes were isolated from Arnebia euchroma, with an average size of 1,010 bp, each gene encoded a 212 amino acids on average. The gene structure and expression of physicochemical properties, subcellular localization, signal peptides, senior structural domains and conservative forecasting, and analysis of A. euchroma were studied comparing with ERF gene gi261363612 of Lithospermum erythrorhizon, and phylogenetic analysis of A. euchroma ERF family was carried out. The results showed the existence of three conserved domains in this family, the senior structure based on random coil and it clustered into CBF/DREB and ERF subfamilies.
Amino Acid Motifs ; Amino Acid Sequence ; Boraginaceae ; genetics ; Cloning, Molecular ; Computational Biology ; Genome, Plant ; genetics ; High-Throughput Nucleotide Sequencing ; Medicine, Chinese Traditional ; Molecular Sequence Data ; Multigene Family ; Phylogeny ; Plant Proteins ; chemistry ; genetics ; Plants, Medicinal ; Protein Structure, Secondary ; Protein Structure, Tertiary ; Sequence Analysis, DNA ; Transcription Factors ; chemistry ; genetics ; Transcriptome

This study aimed to explore the effect and mechanism of acetylalkannin from Arnebia euchroma on the proliferation, migration, and invasion of human melanoma A375 cells. A375 cells were divided into a blank group, and low-, medium-, and high-dose acetylalkannin groups(0.5, 1.0, and 2.0 μmol·L~(-1)). The MTT assay was used to detect cell proliferation. Cell scratch and transwell migration assays were used to detect cell migration ability, and the transwell invasion assay was used to detect cell invasion ability. Western blot was used to detect the protein expression of migration and invasion-related N-cadherin, vimentin, matrix metalloproteina-se-9(MMP-9), and Wnt/β-catenin pathway-related Wnt1, Axin2, glycogen synthase kinase-3β(GSK-3β), phosphorylated GSK-3β(p-GSK-3β), β-catenin, cell cycle protein D_1(cyclin D_1), and p21. Real-time fluorescence-based quantitative polymerase chain reaction(real-time PCR) was used to detect the mRNA expression of E-cadherin, matrix metalloproteinase-2(MMP-2), N-cadherin, vimentin, β-catenin, snail-1, and CD44. MTT results showed that the cell inhibition rates in the acetylalkannin groups significantly increased as compared with that in the blank group(P<0.01). The results of cell scratch and transwell assays showed that compared with the blank group, the acetylalkannin groups showed reduced cell migration and invasion, and migration and invasion rates(P<0.05, P<0.01) and weakened horizontal and vertical migration and invasion abilities. Western blot results showed that compared with the blank group, the high-dose acetylalkannin group showed increased expression of Axin2 protein(P<0.05), and decreased expression of N-cadherin, vimentin, MMP-9, Wnt1, p-GSK-3β, β-catenin, cyclin D_1, and p21 proteins(P<0.05, P<0.01). The expression of GSK-3β protein did not change significantly. PCR results showed that the overall trend of MMP-2, N-cadherin, vimentin, β-catenin, snail-1, and CD44 mRNA expression was down-regulated(P<0.01), and the expression of E-cadherin mRNA increased(P<0.01). Acetylalkannin can inhibit the proliferation, migration, and invasion of human melanoma A375 cells, and its mechanism of action may be related to the regulation of Wnt/β-catenin signaling pathway.
Humans ; Matrix Metalloproteinase 2/metabolism* ; Glycogen Synthase Kinase 3 beta/metabolism* ; beta Catenin/metabolism* ; Vimentin/metabolism* ; Matrix Metalloproteinase 9/metabolism* ; Cell Line, Tumor ; Wnt Signaling Pathway ; Cadherins/genetics* ; Melanoma/genetics* ; Cyclin D/metabolism* ; Cell Proliferation ; Boraginaceae/genetics* ; RNA, Messenger ; Cell Movement

OBJECTIVETo explore the molecular biological mechanism of arnebia root oil in promoting wound surface healing by observing histological change and basic fibroblast growth factor(bFGF) mRNA expression in the wound surface tissues of 2 groups, as well as the wound surface healing rate.

METHODExperimental model of incised-wound was produced on the back of 18 New Zealand albino rabbits. The wound surfaces were randomly divided into two groups, namely, experimental group and control group. The wound surfaces in the experimental group were treated by arnebia root oil and those in control group were treated by petrolatum gauze. Then raw surfaces were evaluated by the techniques of histology, histochemistry and electron microscope and the healing rates of the raw surfaces were compared between the two groups. Content of bFGF and it's mRNA expression in wound surface tissue was also measured by means of Western-blot and RT-PCR.

RESULTThe wound surface healing rate in experimental group was higher than that in control group( P < 0.05). The fibroblast, collagen and blood capillaries were comparatively richer in experimental group as compared with those in control group, and similarly, the expression of bFGF mRNA was also significantly enhanced in the experimental group as compared with control group during the various periods of treatment. In addition, the changes in the expressions of bFGF and it's mRNA paralleled the changes of healing rates in the two groups.

CONCLUSIONthe present results showed that amebia root oil significantly can promote the healing of raw surfaces, which may be mediated by up-regulation of bFGF expression.

Animals ; Boraginaceae ; chemistry ; Female ; Fibroblast Growth Factor 2 ; biosynthesis ; genetics ; Male ; Plant Oils ; isolation & purification ; pharmacology ; Plant Roots ; chemistry ; Plants, Medicinal ; chemistry ; RNA, Messenger ; biosynthesis ; genetics ; Rabbits ; Random Allocation ; Skin ; injuries ; metabolism ; pathology ; Wound Healing ; drug effects ; Wounds and Injuries ; metabolism ; pathology

OBJECTIVETo explore the molecular biological mechanism of Arnebia Root oil (AO) in promoting the recovery of surface of wound by observing basic fibroblast growth factor (bFGF) mRNA expression in the wound tissue and healing rate of the wound.

METHODSPatients in the observed group with their wound treated by AO and those in the control group treated by petrolatum gauze. The wound surface healing rate was estimated and bFGF mRNA expression was observed by RT-PCR.

RESULTSEndogenous bFGF mRNA expression existed in the wound surface of both groups, but its level in the observed group at any time point was obviously higher than that in the control group respectively, with significant difference in comparison of the gray density between the two groups (P < 0.05). The wound surface healing rate kept abreast with bFGF mRNA expression in wound tissues, so it was significantly higher in the observed group than that in the control group (P < 0.05). GAPDH gene, which was taken as a parameter for internal reference, expressed with a certain amount unchanged in different periods of healing (P > 0.05 ).

CONCLUSIONAO shows obviously promotive action on bFGF, an important regulatory factor on wound healing, it might complete the recovery process by stimulating the increase of bFGF.

Adolescent ; Adult ; Boraginaceae ; chemistry ; Drugs, Chinese Herbal ; administration & dosage ; Female ; Fibroblast Growth Factor 2 ; biosynthesis ; genetics ; Humans ; Male ; Middle Aged ; Phytotherapy ; Plant Oils ; administration & dosage ; isolation & purification ; Plant Roots ; chemistry ; RNA, Messenger ; biosynthesis ; genetics ; Wound Healing ; drug effects ; Wounds and Injuries ; drug therapy ; metabolism