Among various technologies used in drug design and discovery, deep learning is still in its infancy. Recently, deep learning approaches have been rapidly developed and applied to address various problems in drug discovery, including generation of virtual compound library, prediction of compound activity, metabolism and toxicity, and prediction of organic synthesis routes. Compared with the traditional machine learning methods, the prediction power of deep learning did not show significant improvement. However, proactively learning and automatically feature extraction bring advantages for deep learning approaches. Compared to first principle-based computational chemistry methods, deep learning can not be generalized because it depends on large-scale and highquality annotated data sets. But its molecular representation with single-atom atomic environment vectors could be useful for computational chemists. As an emerging technology, deep learning, especially the unsupervised learning method that does not rely on large datasets with labels, is gradually improving. It is expected that someday deep learning method will become practical for drug discovery.

Akkermansia muciniphila (A. muciniphila) is an intestinal bacterium that was isolated from human feces in 2004. Its specialization in mucin degradation makes it a key organism that maintains the intestinal mucosal barrier function. A. muciniphila, which is the only representative of the Verrucomicrobia that can be cultured, is relatively easy to be detected in metagenomic analysis. For the past few years, A. muciniphila has quickly attracted the attention of researchers and become a medical and biological hotspot due to the close correlation between its intestinal colonization and the development and progression of various metabolic diseases. This review introduces the biological characteristics and colonization environment of A. muciniphila, and reviews its relationship with host health and disease, especially focusing on the metabolic disease and related mechanism, as well as the factors affecting its colonization in the host, expecting to provide evidence and clues for drug development targeting A. muciniphila.

Protein acetylation is a process of adding an acetyl group to a protein lysine residue with the help of acetyltransferase, which is a pivotal protein post-translational modification linking acetyl-CoA metabolism and cell signal transduction. Recently, the development of mass spectrometry has deepened our understanding of lysine acetylation. Lysine acetylation is involved in many processes such as gene transcription, protein degradation, cellular metabolism, and stress response, which affects biological processes by regulating protein interactions, activity, stability and localization. Protein acetylation is widely happened and plays important regulatory roles in a diversity of human diseases such as metabolic diseases, tumors and cardiovascular diseases. Besides, deacetylase inhibitors have displayed a great potential in the treatment of various diseases especially tumors and metabolic associated diseases. In this review, we summarized the advances and application of acetylation, and discussed the remaining problems in this area.

Amyotrophic lateral sclerosis (ALS) is among the most common type of motor neuron diseases, and its pathogenesis remains unclear. In recent years, our understanding of the genetic basis of ALS has led to the development of various ALS disease models, which allow for screening of ALS-related drugs and treatment methods. This review focuses on the research progress of ALS, summarizes the systems of commonly used experi mental animal models, including transgenic animals, gene knockout approaches and autonomous animal models, points to the problems needing attention in standardized ALS non-clinical research, and proposes the criteria for selection of standardized R&D model.

Based on special scientific facts demonstrated in traditional Chinese medicine (TCM) complex system, this paper proposes a hypothesis of "one output multi-source", discussing the concept, features, structures and the scope. The law of interaction between the integrity and multiple components of TCM complex system was examined. Feasibility, technical methods and evidence supporting the hypothesis have been presented here. We present a basic model of the hypothesis, i.e. artificial neural network (ANN) model. This hypothesis promotes a deeper modern scientific understanding towards the TCM complex system and advancement in research of the material basis. TCM compatibility and quality control will serve as the theoretical foundation for guiding the research on drug combination including chemical, biological and herbal medicines.

The compatibility of traditional Chinese medicines (TCM) includes the compatibility of single herb, effective parts and the effective ingredients of TCM herbs. Compared with the compatibility of Chinese herbal medicine, the compatibility between effective parts and effective ingredients of TCM has become a breakthrough point in the study of compatibility. It provides a new method for precise medication research of TCM, which is bound to become an important driving force in the process of precision medicine research. In this paper, we elabo rate the strategy of the compatibility and proportion in TCM components from five aspects:the compatibility mode of effective components in TCM, the screening method of compatibility of components in TCM, the selection of compatibility component dose, the design method of proportion test and the optimization research of compatibility and proportion.

Enzymes play crucial functional roles in all biological processes. Enzymatic inhibitors can regulate enzyme activity and may become the starting point for drug discovery. Mass spectrometry (MS) has the advantage for rapid qualitative and quantitative analyses of compounds and enzyme reactions, emerging as an important analytical tool in enzyme inhibitor screening assay for drug discovery. This review will highlight recent advances in the inhibitor screening assay using MS and related techniques, including frontier affinity chromatography, immobilized enzyme beads, ultrafiltration, surface plasmon resonance, capillary electrophoresis and microfluidic chips. The existing MS methods for screening enzyme inhibitor were divided into two types:affinity screening and activity screening.

Diabetes is a metabolic disease with an extremely high incidence in China. In parallel with an increased incidence yearly, the population of diabetes is showing a trend towards younger age. Therefore, it is urgent to carry out research on diabetes in order to develop strategy for prevention. In recent years, metabolomics has made significant progress in the study of biomarkers, pathogenesis, early diagnosis and prognosis, and evalua tion of drug efficacy in diabetes. However, limited by metabolomics technology and the complexity of diabetes research, metabolomics in the diabetes research remains challenging. We summarize the progress and prospect the future development of metabolomics in the diabetes research.

Arenaviruses are enveloped RNA viruses, and eight members in this family are known to cause human hemorrhagic fever. Treatments for the viral hemorrhagic fever (VHF) by arenaviruses are very limited. We have identified the first flavone, tangeretin, with broad-spectrum inhibitory activities on VHF-arenaviruses infection by blocking viral entry. In this study, we evaluated thirty-four tangeretin analogues and found 3, 5, 6, 7, 4'-pentamethoxyflavone as a Lassa virus entry inhibitor, with EC₅₀ of 5.2 μmol·L^-1, by blocking the viral fusion pro cess. The compound 3, 5, 6, 7, 4'-pentamethoxyflavone is effective on all known VHF-arenaviruses, with EC₅₀ range of 0.84-10.2 μmol·L^-1. These results suggest that 3, 5, 6, 7, 4'-pentamethoxyflavone is able to serve as a start point for discovery of arenavirus entry inhibitors from flavone natural products.

The purpose of this research is to investigate the effects of acacetin on serum lipid metabolism and atherosclerosis in mice and explore its molecular mechanism. HepG2 cells were treated with different concentrations of acacetin. The expression of LDL receptor (LDLR) and sterol-regulatory element binding protein-2 (SREBP-2) were detected by RT-qPCR and/or Western blot. C57BL/6J mice were given acacetin (50 mg·kg^-1) for 5 weeks by gavage. Serum total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) and triglyceride (TG) were analyzed by an automatic biochemical analyzer. The expression of LDLR or SREBP-2 was detected by Western blot. After 12 weeks of intragastric administration of acacetin (30 mg·kg^-1) in apolipoprotein E knockout (ApoE KO) mice, the serum lipid levels were determined by an automatic biochemical analyzer. The lipid deposition in aortic plaque (en face) and aortic root plaque were stained with oil red O. The expression of LDLR and SREBP-2 were detected by RT-qPCR and/or Western blot. The intestinal content microflora was analyzed by 16S rDNA sequencing (All animal studies were approved by the Animal Experimentation Ethics Committee of Institute of Medicinal Biotechnology, CAMS & PUMC). In vitro results indicated that acacetin significantly up-regulated LDLR mRNA and protein levels, and stimulated LDLR transcription factor SREBP-2 protein expression. As indicated from in vivo studies, compared with control group, acacetin significantly decreased the serum levels of TC and LDL-C in C57BL/6J mice by 34% and 57% (P < 0.01), respectively. Furthermore, mechanic study showed that acacetin significantly increased the protein expression of hepatic LDLR and SREBP-2. Although the results of serum lipid profiles, hepatic LDLR/SREBP-2 expression and area of atherosclerotic lesions in aorta and aortic root in ApoE KO mice showed differences between acacetin and high-fat diet group, the differences did not reach statistical significance. Nevertheless, acacetin exhibited a profound influence on the composition of the intestinal microbiota as indicated by 16s rDNA sequencing analysis. In conclusion, these results demonstrated that acacetin can decrease the serum lipid levels in C57BL/6J mice through up-regulation of hepatic LDLR and SREBP-2, and alter gut microflora in high-fat diet fed Apo KO mice. This study suggests the possibility that acacetin has a potential role in inhibiting the progression of atherosclerosis.

Tuberculosis (TB) is a serious infectious disease caused by Mycobacterium. tuberculosis. In recent years, with the emergence of drug-resistant forms, the development of new anti-tuberculosis drugs is urgently needed. In this study, we used Mycobacterium marinum (M. marinum), which is highly similar to M. tuberculosis, to establish a M. marinum infected-zebrafish model and quantitative PCR (qPCR) method for bacterial count analysis. The results showed that injecting M. marinum into the yolk sac is an efficient and convenient way to infect zebrafish embryos. By counting the survival rate of infected zebrafish and the number of bacteria in zebrafish by ZiehlNeelsen staining, we analyzed the efficacy of isoniazid and rifampicin as anti-tuberculosis drugs and the synergistic effect of drugs. The results suggested that three evaluation methods exhibit good consistency. This study demon strated that zebrafish-M. marinum infection model combined with qPCR analysis is a simple and efficient method for in vivo screening and evaluation of anti-tuberculosis drugs. Animal experiments were carried out in accordance with the provisions for animal ethics in the Regulations on Laboratory Animals of Institute of Medicinal Biotech nology, Chinese Academy of Medical Sciences.

This study aimed to explore the roles of exosomes in doxorubicin-resistance in breast cancer cells. Using breast cancer parental cell line (MCF-7), doxorubicin-resistant cell line (MCF-7/ADR) and sensitive cell line co-cultured with doxorubicin-resistant supernatant (MCF-7/EXO) as models, the effects of doxorubicin on proliferation or apoptosis of MCF-7, MCF-7/EXO and MCF-7/ADR cells were detected by CCK8, and light or fluorescent microscopy. Exosomes in the supernatants of cell culture were extracted by ultracentrifugation, and the quantity of exosomes was determined by transmission electron microscopy, BCA and DiI labeling assay. Expression levels of exosome-specific biomarkers CD63 and Flotillin-1 were detected by Western blot. The uptake of MCF-7/ADR cell-derived exosomes by MCF-7 cells was observed by laser confocal microscopy. Western blot was used to detect the expression levels of multidrug resistance protein ATP-binding cassette subfamily B member 1 (ABCB1) in all three cell strains. Cell proliferation assays showed that IC₅₀ of MCF-7/EXO cells to doxorubicin was 0.83 ±0.09 μmol·L^-1, which was significantly higher than 0.15 ±0.05 μmol·L^-1 (P < 0.01) of MCF-7 cells, suggesting 5.5 times of increase in drug resistance. Apoptosis of MCF-7 cells was induced after doxorubicin treatment (P < 0.001), but MCF-7/EXO cells were not significantly different (P>0.05). Exosome quantification and specific marker detection showed that MCF-7/EXO cells had significantly more exosomes than MCF-7 cells (P < 0.05). PKH67 tracer markers indicated that MCF-7/ADR-derived exosomes could be taken up by MCF-7 cells. Western blot showed that the expression level of ABCB1 protein in MCF-7/EXO cells was significantly higher than that in MCF-7 cells. Taken together, these results indicate that exosomes of doxorubicin-resistant breast cancer cells can transmit drug resistance to sensitive cells, and the underlying mechanism may involve ABCB1 protein transport mediated by exosomes.

The 16S rDNA sequencing method was adopted to study the effects of mulberry leaf flavonoids, polysaccharides and alkaloids on intestinal microflora in db/db diabetic mice. The animal experiment was examined by the Ethics Committee of Nanjing University of Chinese Medicine. Ten db/m mice were control group and forty db/db mice were randomly divided into model group, metformin group, mulberry flavonoid (MF) group, mulberry polysaccharide (MP) group and mulberry alkaloid (MA) group. After intragastric administration for six weeks, fresh feces were collected for detection of intestinal microflora. There were Firmicutes, Bacteroidetes, Proteobacteria, Saccharibacteria, Tenericutes, Deferribacteres, Verrucomicrobia, Cyanobacteria in each group. The results showed that the intestinal microflora of db/db mice changed significantly from phylum level to genus level. The proportion of Firmicutes and Proteobacteria in model group decreased significantly, and the proportion of Bacteroidetes increased. The difference in species abundance distribution between model group and other groups was significant, which indicated that the community distribution was disordered in model group. After administration, the Bacteroidetes, Lachnospiraceae, Roseburia and Desulfovibrio were effectively regulated, especially in the alkaloid group. The difference in species abundance distribution between drug-treated group and blank group also became smaller. It is suggested that the active components of mulberry leaf have the effect of improving the intestinal microflora imbalance in db/db mice.

Based on the concept of network pharmacology, the main nephroprotective components in Erzhi Pill reported in previous studies, were used to predict the targets through the PharmMapper method. Molecular docking was applied to screen for potential targets and biological information annotation databases (DAVID) was used to analyze the molecular function and biological process of the action targets. The Cytoscape software was used to construct the "ingredient-target-pathway" network of Erzhi Pill for renal injury treatment. TTD and GAD database were then applied to screen for the targets of renal disease for building "ingredient-core target" network. We found that 17 major active ingredients of Erzhi Pill regulated 32 targets (including ESR1, ESR2, GCK, MMP3) and affected 6 pathways, such as PI3K-Akt signaling pathway, estrogen signaling pathway and purine metabolism. This study reflected the nature of traditional Chinese medicine as multi-ingredients, multi-targets and multi-path ways, providing new clues for basic science research on the nephroprotective pharmacological mechanism of Erzhi Pill.

A highly sensitive and selective bioluminescent probe for hydrazine (BPH) was designed, synthe sized and evaluated for detection of hydrazine in vitro and in vivo. BPH was designed to include a specific recogni tion group (acetyl) of hydrazine at an appropriate modification site of the optical reporter hydroxyluciferin (Dluciferin), which showed excellent performance both in selectivity and sensitivity to hydrazine. The results showed that the bioluminescent probe BPH developed in this study is an innovative and widely applicable tool for detecting hydrazine in complex natural environment or in animals.

Chemical investigation on the rice culture of Corynespora cassiicola J9, an endophyte inhabiting in Blumea balsamifera (L.) DC. resulted in isolation of eight compounds, including a new depsidone derivative, corynether C (1), and seven analogues, corynether B (2), corynetherlactone A (3), corynether A (4), diaryl ether (5), corynesidone C (6), corynesidone D (7), and corynesidone A (8). Their structures were deduced based on 1D and 2D NMR spectroscopy, and HR-ESI-MS data. All of the isolated compounds were evaluated for inhibitory activities against Lissorhoptrus oryzophilus Kuschel by the leaf spray assay. Unfortunately, none of them showed inhibitory effects.

Snake venom has special pharmacological activities and contains a array of small polypeptides that can antagonize integrins, therefore called disintegrins. Disintegrins can block integrin-dependent platelet aggrega tion, tumor growth, and tumor metastasis. A disintegrin fraction was isolated and purified from the venom of snake Gloydius brevicaudus (GBV). Its physical and chemical properties were characterized, and its biological activities were investigated. The crude venom of GBV were isolated by Superdex 75 gel filtration chromatography. The antiplatelet aggregation activity of the fractions was screened by the Born method. The fraction that shown anti-platelet activity was further purified with Sephadex G-25 gel filtration, DEAE Sepharose Fast Flow ion exchange chroma tography, and Lichrospher C18 reversed-phase chromatography respectively. The purity of the active component was analyzed with SDS-PAGE (Tris-Tricine system) and high-performance liquid-phase chromatography (HPLC), with protein concentration determined by the Bradford method. The molecular weight was evaluated by the gel imaging method and mass spectrometry, and the isoelectric point was measured by disc isoelectric focusing electro phoresis. The protease activity was measured with the Rick method. The phospholipase A activity was determined by the automatic potentiometric titration method. Amino acid sequencing results were subjected to homology comparison using the BLAST program. Seven fractions (Ⅰ-Ⅶ) were isolated from GBV by gel filtration chroma tography on Superdex 75 column. The fraction Ⅳ inhibited the platelet aggregation induced by ADP with molecular weight lower than 10 000 Da, suggesting a disintegrin component. A disintegrin named GBV-Ⅳ 4 was purified from the fraction by Sephadex G-25 gel filtration, DEAE Sepharose Fast Flow ion-exchange and Lichrospher C18 reverse chromatography. It was homogeneous shown as a single band on SDS-polyacrylmide gel electrophoresis (SDS-PAGE, Tris-Tricine system) with molecular weight 8 746 Da as calculated by Image Master VDS system. The isoelectric point of GBV-Ⅳ4 was 6.3 by disc polyacrylamide gel electrophoresis. GBV-Ⅳ4 exhibited no detectable phospholipase A₂ (PLA₂) activity with the pH-stat technique or proteinase activity according to the method of Rick. GBV-Ⅳ4 is composed of 70 amino acids with RGD (Arg-Gly-Asp) active region and a molecular weight of 7 442 Dalton as assayed by Mass Spectrography. Characterization of GBV-Ⅳ4 is consistent with metachain disintegrin (70 amino acid sequence, six pairs of disulfide bond). Retrieved by Genbank, GBV-Ⅳ4 has high homology with other disintegrins. We concluded that GBV-Ⅳ 4 is a novel disintegrin contained RGD. GBV-Ⅳ4 showed dose-dependent inhibition of ADP-or thrombin-induced platelet aggregation with IC₅₀ 0.339 or 0.577 μg·mL^-1 respectively. In conclusion, a new disintegrin derived from the GBV snake venom and named GBV-Ⅳ4 containing RGD tripeptide sequence could inhibit platelet aggregation.

The method for analyzing the interaction between caffeine and human serum albumin (HSA) was established by capillary electrophoresis. Under physiological conditions, the interaction between ligand (caffeine)-receptor (HSA) was studied with frontier-analysis (FA) method, Hummel-Dreyer (HD) method and plug-plug kinetic (PPK) method. The interaction parameters of caffeine-HSA system were obtained using non-linear equation, Scatchard equation and Klotz equation. The results showed that FA, HD and PPK methods were suitable for caffeine-HSA system, among them, HD method was the best, and the Non-linear equation was the best theoretical model to caffeine-HSA system. Interaction parameter tests showed that caffeine-HSA interaction was a single site interaction and the binding stability was moderate. The mechanism of caffeine-HSA interaction has been elucidated, which can provide valuable information for further research of alkaloids.

Nitidine chloride (NC) is a compound with prominent anti-tumor activity. To determine potential cardiotoxicity of NC, this study was designed to investigate the distribution of NC in rat heart and the underlying mechanism. The animal studies were approved by Institutional Animal Care and Use Committee of Zhejiang University Medical Center (2015-380-01) and complied with the standards of animal welfare in China. At 0.25, 0.5 and 2 h after a single intravenous injection (iv) of 5 mg·kg^-1 NC, the concentrations of NC in rat heart were 47.7, 71.1 and 63.2 μg·g^-1 respectively, which were 576, 1 352 and 1 212 folds of that in plasma. This study also revealed that the NC concentration in heart was 458.5 μg·g^-1 (7 336 folds of that in plasma) at 2 h after the last dose in rats, after daily iv administration of NC at 5 mg·kg^-1·day^-1 for successive 20 days. Further studies showed that the accumulations of NC in MDCK-hOCT1 and MDCK-hOCT3 cells were 16.1 and 4.99 folds higher than that of the mock cells, respectively. There is no significant difference between the accumulations of NC in MDCK cells transfected with hOCTN1, hOCTN2 or hPMAT and the mock cells. Additionally, quinidine, L-tetrahydropalmatine and Decynium 22, the inhibitors of OCTs, clearly reduced the accumulations of NC in primary cardiomyocytes and cardiac fibroblasts from neonatal rats. MTT assay showed that the LC₅₀ of NC on cardiomyocytes and cardiac fibroblasts were 10.9 and 10.4 μmol·L^-1, respectively. Moreover, treatment of the primary cardiomyocytes and cardiac fibroblasts with NC (1~15 μmol·L^-1) for 48 h resulted in significantly increased LDH enzyme leakage. These results indicated that NC can be highly accumulated in the heart, and accumulation is mediated by OCT1 and OCT3, but not by OCTN1, OCTN2 and PMAT. The accumulated NC has potential cytotoxicity as shown in the results from primary cardiomyocytes and cardiac fibroblasts.

The study was designed to synthesize a novel dendritic copolymer composed of polyamidoamine dendrimer G0 as the inner core and poly(L-glutamic acid) grafted low molecular weight polyethylenimine (PGLP) as surrounding arms for gene delivery vector. The molecular structure of PGLP was confirmed by ¹H NMR (proton nuclear magnetic resonance spectroscopy). The DNA combination capability of PGLP was examined by gel retarda tion electrophoresis. The particle sizes and zeta potentials of PGLP/pDNA complexes were determined by dynamic light scattering (DLS). The cytotoxicity of PGLP was evaluated by Cell Counting Kit-8 (CCK-8) and hemolysis assays, which was approved by Research Ethics Committee of the First Affiliated Hospital of Nanchang University. The in vitro transfection efficiency of PGLP was measured by a flow cytometry. The results of physicochemical properties suggested that PGLP could self-assemble with DNA to form complexes with average particle sizes of about 105-200 nm and zeta potentials of about +10-+28 mV, which could protect DNA from serum degradation. The results of biological properties suggested that PGLP showed more higher transfection efficiencies but lower cytotoxicity than PEI 25K or Lipofectamine 2000 in various cell lines (HEK 293T, HeLa, BEL 7402, RASMC). Importantly, it was found that PGLP/pDNA complexes at w/w=8 showed more strong serum-resistant capacity than PEI 25K/pDNA complexes. Therefore, PGLP is a promising candidate vector for gene delivery.

Photodynamic therapy (PDT) is one of the new approaches for cancer treatment with high efficacy. However, applications of current photosensitizers are restricted to skin and superficial tumor due to poor in vivo targeting ability, poor water solubility and short wavelength excitement, which limits penetration therefore thera peutic depth. Here, a biodegradable polymeric micelle, methoxy poly(ethylene glycol)-polylactide copolymer (mPEG-PDLLA), is employed as drug delivery system to co-encapsulate strong two-photon absorption compound (L_TPA) and photosensitizers. This delivery system is designed to target tumor passively, resulting in near infrared light with an approximately 808 nm wavelength becoming able to indirectly excite photosensitizers through fluores cence resonance energy transfer. Tumor cells and microvessels could be damaged by the generated singlet oxygen. The average size of drug loaded micelles was approximately 55 nm and showed a spherical shape. Both com pounds could be released simultaneously from micelles under either weak acid and neutral pH conditions. Reactive oxygen species was produced intracellularly during two-photon PDT process and induced cell apoptosis/necrosis, which was quantified by Annexin-V/FITC assays. Time-dependent ex vivo organ distribution and in vivo anticancer efficacy results suggested that the drug carriers could accumulate in tumors and suppress tumor growth by twophoton PDT. All animals experiments were performed in line with national regulations and approved by the Animal Experiments Ethical Committee of College of Pharmaceutical Sciences, Southwest University. In summary, we have employed two-photon PDT for breast cancer treatment successfully in a mouse model and have demonstrated the significance of delivery system in such therapeutics.

To accurately discriminate Stellariae Radix from its adulterants, four leading candidate DNA barcoding markers were evaluated. Sixty samples including Stellariae Radix and its adulterants have been newly collected and their total genomic DNA was extracted. Four DNA barcoding markers ITS, rbcL, psbA-trnH and matK were amplified and sequenced. Their sequence characteristic analyses, Kimura-2-parameter (K2P) distance calculation and Neighbor-joining (NJ) phylogenetic tree constructions were accomplished using the MEGA 7.0 software. DNA Barcoding gaps of the four DNA barcoding markers were estimated by the distributions of interand intra-sequence specific variations. Species identification efficiency was calculated using the BLAST method. The results showed that ITS had the highest (95.2%) while matK demonstrated the lowest (75%) PCR and sequencing efficiency. The length range of the four markers were in the ranger of 211-797 bp, and the G+C content of ITS was highest (54.35%). The identification efficiency of matK and ITS was 92% and 90% respectively. Barcoding gap could be found in ITS sequences. The NJ phylogenetic tree constructed using ITS sequences showed that samples of Stellariae Radix were separately formed into one clade, and samples of adulterants like Stellaria bistyla were clearly belong to different branches from Stellariae Radix, whereas NJ trees constructed using psbA-trnH, rbcL and matK could not differentiate Stellariae Radix from its adulterants. Therefore, ITS regions as DNA barcodes can stably and accurately distinguished Stellariae Radix from its adulterants, and provide a new technique for modern identification of Stellariae Radix.

The roots and flowers of Gentiana waltonii and Gentiana robusta are used as Tibetan herb Jie-Ji in traditional Tibetan medicine, with iridoids as the main active ingredient and index components. To study the pathway of iridoid biosynthesis, roots, stems, leaves and flowers of G. waltonii and G. robusta were subjected to a high-throughput transcriptomic sequencing analysis by Illumina HiseqXTen. After removing insignificant reads and de novo splicing, 79 455 and 78 466 unigenes were obtained from G. waltonii and G. robusta respectively, with average length as 834 bp and 862 bp. The unigene GO functions could be divided into three categories of 65 branches. The unigenes were aligned in KOG database and were classified into 25 classes according to function. In KEGG database, 315 and 340 unigenes of G. waltonii and G. robusta were implicated in 20 standard secondary metabolic pathways, respectively. Furthermore, 80 and 57 unigenes of the two species were analyzed to encode 24 key enzymes in the pathway related to iridoid biosynthesis. There were differences in gene expression among different organs. Based on sequence data, significant amounts of SSRs, SNPs and InDels were detected in each dataset. This study provides a platform for further development of molecular markers, excavation of functional genes, and research into metabolic pathways and their regulatory mechanism within G. waltonii and G. robusta.