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Gentiana macrophylla is one of Chinese herbal medicines in which 4 kinds of iridoids or secoiridoids, such as loganic acid, sweroside, swertiamarin, and gentiopicroside, are identified as the dominant medicinal secondary metabolites. WRKY, as a large family of transcription factors (TFs), plays an important role in the synthesis of secondary metabolites in plants. Therefore, WRKY genes involved in the biosynthesis of secoiridoids in G. macrophylla were systematically studied. First, a comprehensive genome-wide analysis was performed, and 42 GmWRKY genes were identified, which were unevenly distributed in 12 chromosomes. Accordingly, gene structure, collinearity, sequence alignment, phylogenetic, conserved motif and promoter analyses were performed, and the GmWRKY proteins were divided into three subfamilies based on phylogenetic and multiple sequence alignment analyses. Moreover, the enzyme-encoding genes of the secoiridoid biosynthesis pathway and their promoters were then analysed, and the contents of the four secoiridoids were determined in different tissues. Accordingly, correlation analysis was performed using Pearson′s correlation coefficient to construct WRKY gene-enzyme-encoding genes and WRKY gene–metabolite networks. Meanwhile, G. macrophylla seedlings were treated with methyl jasmonate (MeJA) to detect the dynamic change trend of GmWRKYs, biosynthetic genes, and medicinal ingredient accumulation. Thus, a total of 12 GmWRKYs were identified to be involved in the biosynthesis of secoiridoids, of which 8 (GmWRKY1, 6, 12, 17, 33, 34, 38 and 39) were found to regulate the synthesis of gentiopicroside, and 4 (GmWRKY7, 14, 26 and 41) were found to regulate the synthesis of loganic acid. Taken together, this study systematically identified WRKY transcription factors related to the biosynthesis of secoiridoids in G. macrophylla, which could be used as a cue for further investigation of WRKY gene functions in secondary metabolite accumulation. [ABSTRACT FROM AUTHOR]

Gentiana macrophylla is a perennial herb in the Gentianaceae family, whose dried roots are used in traditional Chinese medicine. Here, we assembled a chromosome-level genome of G. macrophylla using a combination of Nanopore, Illumina, and Hi-C scaffolding approaches. The final genome size was ~1.79 Gb (contig N50 = 720.804 kb), and 98.89% of the genome sequences were anchored on 13 pseudochromosomes (scaffold N50 = 122.73 Mb). The genome contained 55,337 protein-coding genes, and 73.47% of the assemblies were repetitive sequences. Genome evolution analysis indicated that G. macrophylla underwent two rounds of whole-genome duplication after the core eudicot γ genome triplication event. We further identified candidate genes related to the biosynthesis of iridoids, and the corresponding gene families mostly expanded in G. macrophylla. In addition, we found that root-specific genes are enriched in pathways involved in defense responses, which may greatly improve the biological adaptability of G. macrophylla. Phylogenomic analyses showed a sister relationship of asterids and rosids, and all Gentianales species formed a monophyletic group. Our study contributes to the understanding of genome evolution and active component biosynthesis in G. macrophylla and provides important genomic resource for the genetic improvement and breeding of G. macrophylla. [ABSTRACT FROM AUTHOR]

Gentiana macrophylla Pall. (G. macrophylla)—a member of the family Gentianaceae—is a well-known traditional Chinese medical herb. Iridoids are the main active components of G. macrophylla, which has a wide range of pharmacological activities such as dispelling wind, eliminating dampness, clearing heat and asthenic fever, hepatoprotective and choleretic actions, and other medicinal effects. In this study, a total of 67,048 unigenes were obtained by transcriptomic sequencing analysis of G. macrophylla. A BLAST analysis showed that 48.21%, 33.66%, 46.32%, and 32.62% of unigenes were identified in the NR, Swiss-Prot, eggNOG, and KEGG databases, respectively. Twenty-five key enzymes were identified in the iridoid biosynthesis pathway. Most of the upregulated unigenes were enriched in flowers and leaves. The trustworthiness of the transcriptomic data was validated by real-time quantitative PCR (qRT-PCR). A total of 22 chemical constituents were identified by ultra-high performance liquid chromatography-quadrupole-electrostatic field Orbitrap mass spectrometry (UPLC-Q-Exactive MS), including 10 iridoids. A correlation analysis showed that the expression of 7-DLH and SLS was closely related to iridoids. The expression of 7-DLH and SLS was higher in flowers, indicating that flowers are important for iridoid biosynthesis in G. macrophylla. [ABSTRACT FROM AUTHOR]

Efficient high-performance countercurrent chromatography methods were developed to isolate five typical compounds from the extracts of Gentiana macrophylla. n-Butanol-soluble extract of G. macrophylla contained three hydrophilic iridoids, loganic acid ( 1), swertiamarin ( 2) and gentiopicroside ( 3), and a chromene derivative, macrophylloside D ( 4) which were successfully isolated by flow rate gradient (1.5 mL/min in 0-60 min, 5.0 mL/min in 60-120 min), and consecutive flow rate gradient HPCCC using n-butanol/0.1% aqueous trifluoroacetic acid (1:1, v/v, normal phase mode) system. The yields of 1- 4 were 22, 16, 122, and 6 mg, respectively, with purities over 97% in a flow rate gradient high-performance countercurrent chromatography, and consecutive flow rate gradient high-performance countercurrent chromatography gave 1, 2, 3 (54, 41, 348 mg, respectively, purities over 97%) and 4 (13 mg, purity at 95%) from 750 mg of sample. The main compound in methylene chloride soluble extract, 2-methoxyanofinic acid, was successfully separated by n-hexane/ethyl acetate/methanol/water (4:6:4:6, v/v/v/v, flow-rate: 4 mL/min, reversed phase mode) condition. The structures of five isolates were elucidated by 1H, 13C NMR and ESI-Q-TOF-MS spectroscopic data which were compared with previously reported values. [ABSTRACT FROM AUTHOR]

Real time quantitative PCR (RT-qPCR or qPCR) has been extensively applied for analyzing gene expression because of its accuracy, sensitivity, and high throughput. However, the unsuitable choice of reference gene(s) can lead to a misinterpretation of results. We evaluated the stability of 10 candidates - five traditional housekeeping genes (UBC21, GAPC2, EF-1α4, UBQ10, and UBC10) and five novel genes (SAND1, FBOX, PTB1, ARP, and Expressed1) - using the transcriptome data of Gentiana macrophylla. Common statistical algorithms ΔCt, GeNorm, NormFinder, and BestKeeper were run with samples collected from plants under various experimental conditions. For normalizing expression levels from tissues at different developmental stages, GAPC2 and UBC21 had the highest rankings. Both SAND1 and GAPC2 proved to be the optimal reference genes for roots from plants exposed to abiotic stresses while EF-1α4 and SAND1 were optimal when examining expression data from the leaves of stressed plants. Based on a comprehensive ranking of stability under different experimental conditions, we recommend that SAND1 and EF-1α4 are the most suitable overall. In this study, to find a suitable reference gene and its real-time PCR assay for G. macrophylla DNA content quantification, we evaluated three target genes including WRKY30, G10H, and SLS, through qualitative and absolute quantitative PCR with leaves under elicitors stressed experimental conditions. Arbitrary use of reference genes without previous evaluation can lead to a misinterpretation of the data. Our results will benefit future research on the expression of genes related to secoiridoid biosynthesis in this species under different experimental conditions. [ABSTRACT FROM AUTHOR]

This paper elaborates on the seedling technology, field cultivation technology, field management technology, seed harvesting and seed processing technology in the propagation process of Gentiana macrophylla to provide technical support for artificial breeding of G. macrophylla. [ABSTRACT FROM AUTHOR]

As a result of the wide distribution of herbal teas the data on nutritional characterisation, chemical profile and biological activity of these products are required. The decoctions of Gentiana algida, G. decumbens, G. macrophylla and G. triflora herb teas were nutritionally characterized with respect to their macronutrients, demonstrating the predominance of polysaccharides and low lipid content. Gentian decoctions were also submitted to a microcolumn RP-HPLC-UV analysis of phytochemicals demonstrating a high content of iridoids (177.18-641.04 μg/mL) and flavonoids (89.15-405.71 μg/mL). Additionally, mangiferin was detected in samples of G. triflora tea (19.89 μg/mL). Five free sugars (fructose, glucose, sucrose, gentiobiose, gentianose) were identified in all gentian teas studied, as well as six organic acids (malic, citric, tartaric, oxalic, succinic, quinic). Pectic polysaccharides with a high content of rhamnogalacturonans and arabinogalactans were also identified and characterized in gentian decoctions for the first time. Gentian tea decoctions and their specific compounds (gentiopicroside, loganic acid-6'-O-β-D-glucoside, isoorientin, isoorientin-4'-O-β-D-glucoside, mangiferin, water-soluble polysaccharides) showed a promising antimicrobial, anti-inflammatory and antioxidant potentials. Evidences obtained indicate the prospective use of gentian herb teas as food products and medicines. [ABSTRACT FROM AUTHOR]

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Some Gentiana species have been used by the nomadic people of Siberia as bitter teas or appetizers to eliminate digestive disorders (dyspepsia, heartburn, nausea, etc.). We studied the most frequently used gentians: Gentiana algida, G. decumbens, G. macrophylla and G. triflora. The aim of the present study was to evaluate the phytochemical features and gastrostimulatnt activity of these four gentian herbs. Five iridoids, seven flavones and mangiferin were detected in gentian herbs after analysis by microcolumn-RP-HPLC-UV-ESI-MS. A componential phytochemical profile of the G. decumbens herb is presented for the first time, as well as information about distinct phytochemicals found in gentian herbs. HPLC quantification of the specific compounds of gentian herbs demonstrated the high content of iridoids (24.73-73.53 mg/g) and flavonoids (12.92-78.14 mg/g). The results of biological activity evaluation of four gentian decoctions demonstrated their good ability to stimulate acid-, enzyme- and mucin-forming functions of the stomach attributed to mostly by iridoids and flavonoids. In general, it can be claimed that the gentian decoctions can be used as effective and safe appetizers and are also a good source of biologically active agents. [ABSTRACT FROM AUTHOR]

The dried root of Gentiana macrophylla is a well-known traditional Chinese herbal medicine for treating jaundice, hepatitis, and stomachic and choleretic ailments. However, natural sources are now in short supply. A lack of information about its genetic background has been a great hindrance to producing its active constituents via genetic engineering. We performed RNA-seq to obtain 42,918 unigenes (average length = 667 bp) in its transcriptome. Of these, 32,141 (74.89 %) were annotated and 2,339 unigenes were assigned to secondary-metabolite pathways. In all, 114 putative unigenes involved in secoiridoid biosynthesis were identified in our transcriptome library. A Blast X search against the Arabidopsis gene regulatory information server showed that 4,413 unigenes are homologous to transcription factor genes from Arabidopsis. Organ-specific genes and candidate gene expression profiles were also investigated with digital gene expression technology. Quantitative PCR was used to verify the expression patterns of several novel transcripts involved in secoiridoid biosynthesis. Our results not only enrich the gene resource but will also benefit research into the molecular genetics and functional genomics of this species. [ABSTRACT FROM AUTHOR]

A novel dimeric secoiridoid glucoside, gentimacroside (1), and six known compounds (2-7) were isolated from the roots of Gentiana macrophylla Pall. Their structures were established on the basis of spectroscopic analysis, especially by means of 1D-, 2D-NMR and HRESIMS analyses. [ABSTRACT FROM AUTHOR]

The leaf explants from the eight-week-old Gentiana macrophylla Pall sterile seedlings were precultured for three days on Murashige and Skoog (MS) agar medium with 1 mg/l of 6-benzyladenine, and then incubated in MS liquid medium containing Agrobacterium rhizogenes R1000 for 2 h with shaking. After 10 days of culture on hormone-free MS agar medium with 500 mg/l cefotaxime under illumination of 300 μmol/(m2 s) with a 16-h photoperiod at 24°C, up to 18.3% of the infected explants produced typical hairy roots (HRs). Finally, three stable HR lines with no morphologically visible differences were obtained. The HR lines could vigorously grow and propagate on/in hormone-free half-strength MS solid/liquid medium. Moreover, they changed from white to green when moved from darkness to the light and spontaneously produced calli, which were able later to produce adventitious buds. PCR analysis revealed that three HR lines, which were undergone to continuous subculturing over two years, still contained the rolC gene from the A. rhizogenes Ri plasmid. HPLC revealed the HR line possessing a certain capacity of synthesizing gentiopicroside (0.11 mg/g dry wt). [ABSTRACT FROM AUTHOR]

Gentiana macrophyllais a perennial medicinal plant in the family Gentianaceae. Natural sources forG. macrophyllaplants are being exhausted due to over-collection and need urgent conservation. The complete chloroplast DNA sequence ofG. macrophylla(GenBank accession number: KY856959) was determined in our study. The size of chloroplast genome ofG. macrophyllais 149,916 bp, including a large single-copy (LSC) region of 82,911 bp and a small single-copy (SSC) region of 17,095 bp separated by a pair of identical inverted repeat regions (IRs) of 24,955 bp each. A total of 130 genes were successfully annotated containing 86 protein-coding genes, 36 tRNA genes and 8 rRNA genes. The GC content is 37.7%. 33 chloroplast simple sequence repeats (cpSSRs) ofGentiana macrophyllawere detected, in which 32 of them are mononucleotide repeats. Phylogenomic analysis strongly supported the close relationship ofG. macrophyllaand other 5 species in Gentiana. [ABSTRACT FROM PUBLISHER]