Distribution and diversity of endophytic fungi in Gentiana rigescens and cytotoxic activities / 中草药·英文版

Objective: In the present study, Gentiana rigescens was screened for fungi communities to clarify their diversity and community assemblage in hosts. Meanwhile, the identification and activity assays of the strains were also conducted. Methods: By culture-dependent (endophytic fungi isolations from plant sections) and culture-independent (metagenomic library and cloning from plant sections) techniques, fungi communities were studied. The metagenomic library was generated using direct DNA isolation of whole plants, plant radixes, plant stems, plant leaves, plant flowers and soils around the plant. Meanwhile, endophytes were isolated from all parts of G. rigescens plants. After fermentation of the fungi isolations, all the isolates were evaluated for their cytotoxicity against four kinds of human cancer cell lines (HCT116, BEL7404, A549, MDA-MB-231). Results: Eventually, 200 strains were isolated and 103 strains were further identified through the internal transcribed spacer (ITS, ITS1 and ITS2 regions) sequence by using the universal primers ITS5 and ITS4. A total of 59,106 fungal sequences corresponding to 374 putative operational taxonomic units (OTU) were identified by 454 pyrosequencing. Through 454 pyrosequencing, the main fungal genera were Sebacina, Botrytis, Mycosphaerella, Boletus and Gibberella, and the major fungal genera which were directly isolated were Aspergillus, Fusarium, Penicillium and Alternaria. Activity assays showed strains 5–26 (Aspergillus sp.) and 6–2 (Fusarium avenaceum) had the outstanding cytotoxicity to all the tested cell lines with IC50 values <5 μg/mL. Conclusion: This study revealed the abundance of endogenetic fungal resources and a variety of genetic information in G. rigescens by high-throughput 454 sequencing technology and fungi isolation methods. Activity assays indicated that endophytes were a promising natural source of potential anticancer agents.

Efficient induction system of adventitious shoot and plantlet regeneration in Gentiana rigescens / 中草药

Objective: An efficient and stable regeneration system was developed to select the optimal protocol for plant regeneration with various explants of Gentiana rigescens. Methods: The study was designed as a L9(34) orthogonal experiment to explore the effects of different types and concentration of plant growth regulator combinations on adventitious shoot formation and plant regeneration, using different explants maintained on Murashige and Skoog (MS) medium after a single factor pre-experiment had been conducted. Results: Stem with buds was the optimal explant for indirect organogenesis with a frequency of callus induction of up to 97.73% obtained on MS + 6-BA 1.5 mg/L + NAA 1.0 mg/L + KT 0.05 mg/L after 10 dwith the axillary shoots starting to germinate along with the emergence of callus with a strong capacity of differentiation from the base; The callus started to generate green multiple shoots with a 100% rate after 15 d and a coefficient of differentiation in the clump shoot of 18.65. The multiplication coefficient was up to 63.58. However, the stem tip was the best explant for direct organogenesis with a propagation coefficient of up to 44.36 cultured on MS + 6-BA 0.5 mg/L + NAA 1.0 mg/L + KT 0.5 mg/L 30 days later. The plantlet derived from two explants was too weak for rooting and the weak multiplication plantlet was maintained on MS medium supplemented with 6-BA 1.5 mg/L, NAA 1.0 mg/L, KT 0.05 mg/L, and PP333 10 mg/L to rejuvenation for 60 d. The regeneration plant with 100% rooting rate was obtained by culturing on 1/2 MS + 6-BA 0.1 mg/L + NAA 2.0 mg/L for 45 d and more than 95% of plantlets survived after transplanting. Conclusion: The current study established a rapid propagation system which is helpful to protect the wild resources and high quality seedling propagation of G. rigescens, meanwhile providing scientific evidence for the research on genetic transformation.

Cloning and bioinformatics analysis of iridoid oxidase gene in Gentiana rigescens / 中草药

Objective To obtain the key enzyme gene involving in the monoterpenoid biosynthesis pathway, an iridoid oxidase gene (GrIDO) and its promoter were cloned from the leaves of Gentiana rigescens, and its bioinformatics analysis were also performed. Methods The gene specific primers were designed according to the GrIDO gene of transcriptome in G. rigescens. The open reading frame (ORF) of GrIDO gene was cloned by RT-PCR method. The bioinformation of GrIDO gene was analyzed by online softwares. Meanwhile, the gene specific primers were designed according to the cloned GrIDO gene, and the promoter of GrIDO gene was amplified by PCR method. Its sequence analysis was also performed. Results The length of GrIDO gene ORF (GenBank accession number KP722034) was 1 557 bp, which encoded a protein with 518 amino acids. Its relative molecular weight was 58 920 with the theoretical isoelectric point of 8.40. GrIDO was the member of cytochrome P450 superfamily and may localize in chloroplast. GrIDO was a hydrophilic stable protein without signal peptide and composed of mainly α-helix (51.07%) and loops (42.69%). GrIDO protein had a high similarity with CrIDO of Catharanthus roseus (85.83%) and their genetic relationship was close. The cloned GrIDO promoter had a length of 720 bp (GenBank accession number KT428570), which possessed cis-regulatory elements TATA-box and CAAT-box, and had cis-acting elements involved in the abscisic acid and MeJA responsiveness, part of a light responsive element and MYB transcription factor binding site. Conclusion The expression of GrIDO gene were regulated by multifactors. The GrIDO gene and its promoter were cloned from G. rigescens. This study will lay foundations for the functional research of GrIDO gene in monoterpenoid biosynthesis pathway.

Molecular cloning, differential expression, and bioinformatics analysis of G10H gene in Gentiana rigescens / 中草药

Objective: To clone the geraniol-10-hydroxylase (G10H) gene from Gentiana rigescens and analyze the gene expression. Methods: The cDNA sequence of G10H was obtained from G. rigescens using PCR technique. The physical and chemical properties, secondary structure and tertiary structure of G10H protein were forecasted and analyzed using related software. The expression of G10H gene was detected using real-time PCR in roots, stems, and leaves of G. rigescens. Results: The cloned G10H gene was 1 400 bp including 1 248 bp open reading frame and encoding a predicted protein of 415 amino acids. Bioinformatics predicted that the gene encoding protein molecular formula was C2131H3390N586O615S17, the isoelectric point was 7.62, the instability coefficient was 44.20, and the hydrophobic coefficient was -0.245. RT-PCR showed that G10H gene expressed in all organs. And the highest expression level was found in roots, while the lowest in the stems. Conclusion: This G10H gene is cloned from G. rigescens for the first time. The results provide a foundation for exploring the mechanism of gentiopicroside biosynthesis in G. rigescens.

HPLC characteristic fingerprints of Gentiana scabra Bge. and Gentiana rigescens Franch. based on the polarity orientation technology / 中国药学杂志

OBJECTIVE: To establish the HPLC characteristic fingerprints of Gentiana scabra Bge. and G. rigescens Franch. METHODS: The characteristic fingerprints were established based on polarity orientation technology. Similarity analysis, cluster analysis, and principal component analysis were used to evaluate the fingerprints. RESULTS: The HPLC fingerprints of ethyl acetate fraction from the water extract of G. scabra and G. rigescens showed obvious characteristics. The overall similarity of 21 batches of samples was 0.29-0.95. The overall similarity of G. scabra was 0.82-0.99 except S4. And that of G. rigescens was 0.75-0.99 among which 10 batches were greater than 0.90. In the cluster analysis, the samples were divided into three clusters: S4, G. scabra, and G. rigescens. In the principal component analysis, 21 batches of G. scabra. and G. rigescens could be clearly divided into two categories, while S4 was far away from the other samples of G. scabra. CONCLUSION: G scabra and G. rigescens can be identified effectively by the characteristic fingerprints. The method can be used for the quality control of Radix Gentianae.

Chemoetrics analysis of HPLC fingerprint of Gentiana rigescens from different multiple cropping systems and application in quality evaluation / 中国药学杂志

OBJECTIVE: To establish the HPLC fingerprint of Gentiana rigescens Franch. from different cultivation systems for the quality evaluation of the medicinal materials in combination with chemometrics analysis. METHODS: Four bioactive compounds, ie loganic acid, swertiamarin, gentiopicroside, and sweroside, were qualitatively and quantitatively analyzed. The chromatographic analysis was performed on a Shim-pack VP-ODS C18 column(4.6 mm×150 mm, 5 μm) at a temperature of 30℃. Linear gradient elution was used with mobile phase consisting of water with 0.1% formic acid(A) and acetonitrile(B). The flow rate was 1.00 mL·min-1. The injection volum was 10 μL. The detection wavelength was set at 241 nm. Chemometrics methods including hierarchical cluster, partial least squares discriminant analysis, and variable importance were used for this study. RESULTS: The calibration curves of the four compounds were linear in the range of 10-4000 μg·mL-1. The coefficients ranged from 0.9992 to 0.9999. The precision RSDs of the four compounds were 0.87%, 2.06%, 0.90% and 2.2%, respectively. The repeatability RSDs were 0.97%, 1.9%, 2.0% and 2.2%, respectively. The stability RSDs were lower than 2.2%. The recoveries of the four compounds varied from 97.12% to 103.81% with RSDs from 0.71% to 2.0%. The similarity analysis showed similarity values over 0.970 for most samples. The constituents of 70 samples collected from different cultivation systems were similar. However, the contents of those compounds varied significantly in the following order; gentiopicroside>swertiamarin>loganic acid>sweroside. The chemometrics characteristics of gentiopicroside was significantly different from the other compounds. The total content of the four compounds was the highest in the samples cultivated under Alnus nepalensis D. Don. The samples cultivated under Juglans regia Linn, had the highest content of loganic acid. The samples cultivated under Eucalyptus robusta Smith, had the highest content of swertiamarin. The samples cultivated under Cunninghamia lanceolata (Lamb.) Hook, had the highest content of sweroside. The results of partial least squares discriminant analysis and variable importance for the study showed that gentiopicroside (VIP=1.513) and swertiamarin (VIP=1.208) were important variables for discrimination of cultivation system. The compounds with retension time of more than 10 min were important for discrimination of cultivation condition too. CONCLUSION: HPLC fingerprint combined with contents of main bioactive compounds could provide method and theoretical basis for the quality evaluation of G. rigescens Franch. from different multiple cropping systems.

Quality evaluation and quality equivalence of Gentiana rigescens Franch. from different populations / 中国药学杂志

OBJECTIVE: To establish evaluation methods of the quality and quality equivalence of Gentiana rigescens Franch. from different populations. METHODS: The contents of the active ingredients, gentiopicroside, swertiamarin and sweroside in the roots of Gentiana rigescens Franch., were determined by HPLC, and the HPLC fingerprint was established. The quality and quality e-quivalence were evaluated by One-way ANOVA, cluster analysis and principal component analysis (PCA). RESULTS: The overall quality of Gentiana rigescens Franch. from different populations in Dali Yunnan was better. The content of gentiopicroside in the sample from Weishan population was lower than those in the other populations, while it was more than twice the national standard. There was significant difference (P<0.01) in the contents of gentiopicroside, swertiamarin, and sweroside in the roots of Gentiana rigescens Franch. from different populations. The quality equivalence of Gentiana rigescens Franch. from different populations was analyzed, and 11 common peaks in HPLC fingerprint were selected to evaluate the similarities of the crude drugs. The similarities of the characteristic peaks were all above 0.988. The three characteristic peaks of gentiopicroside, swertiamarin and sweroside which were related to the activity of Gentiana rigescens Franch were identified, and the qualities of different populations were basically equivalent. The total contents of gentiopicroside, swertiamarin and sweroside of Gentiana rigescens Franch. from different populations were similar as shown by cluster analysis and PCA of HPLC fingerprint. CONCLUSION: Jianchuan and Heqing polulations are quality germplasm populations according to the contents of effective components and HPLC fingerprint. The evaluation methods of quality and quality equivalence of Gentiana rigescens Franch. from different populations are established, which would provide the theoretical basis for further filtering of quality germplasm resources of Gentiana rigescens Franch. from wild populations.

Cloning, sequence analysis, and prokaryotic expression of GrCYP450-17 gene in Gentiana rigescens / 中草药

Objective: To obtain the key enzyme gene GrCYP450 involving in the terpenoid biosynthesis, a CYP450-17 gene was cloned from young leaves of Gentiana rigescens, and its sequence analysis and prokaryotic expression were performed. Methods: According to the GrCYP450-17 gene sequence of transcriptome of triennial G. rigescens, a pair of primers were designed, and the ORF (Open reading frame)of cDNA sequences was obtained by RT-PCR. Then TA cloning, sequencing, and sequence analysis were performed. Prokaryotic expression vector pGEX-4T-1-GrCYP450-17 was constructed and transformed into E. coli Rosetta (DE3) for the expression under the induction of Isopropyl β-D-1-Thiogalactopyranoside (IPTG.). Results: The ORF of GrCYP450-17 has a length of 1 545 bp coding for 514 amino acids. Sequence analysis showed that GrCYP450-17 was the member of CYP714 family. Results of phylogenic analysis showed that GrCYP450-17 was close to SlCYP450 of tomato. The SDS-PAGE results showed that the expressed proteins were consistent with the anticipated size. Conclusion: The GrCYP450-17 gene is successfully cloned, and the stable prokaryotic expression system is established. This study will provide a foundation for the further purification, structural and functional researches of GrCYP450-17 protein.

Cloning, sequence analysis, and prokaryotic expression of GrGPPS gene in Gentiana rigescens / 中草药

Objective: To obtain the indispensable key enzyme involved in the monoterpene biosynthesis, the geranyl diphosphate synthase gene GrGPPS was cloned from Gentiana rigescens, and its sequence analysis and prokaryotic expression were performed. Methods: According to the GrGPPS gene sequence of transcriptome of triennial G. rigescens, a pair of specific primers was designed, and the full length of cDNA sequences was obtained by RT-PCR. Then TA cloning, sequencing and sequence analysing were performed. Prokaryotic expression vector pGEX-T-1-GrGPPS was constructed and transformed into Escherichia coli Rosetta for expression under 37°C and induced by 1 mmol/L IPTG. Results: The GrGPPS cDNA had a length of 1107 bp coding for 369 amino acids. Sequence analysis showed that GrGPPS was the member of "short-chain prenyltransferases" super family. Results of phylogenic analysis showed that GrGPPS was at the same evolutionary branch with AmGPPS. The SDS-PAGE results displayed that the expressed proteins were consistent with the anticipated size. Conclusion: The GrGPPS gene is cloned from G. rigescens, and the stable prokaryotic expression system of pGEX-T-1-GrGPPS is constructed. This work will provide a foundation for further purification, structure, and functional research of GrGPPS protein.