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Background: To determinethe involvement of hypoxia inducible factor1α (HIF) pathway inmetastasis of gastric cancer (GC). Method: It was determined that the expression of HIF and annexin A1 (ANXA1) in human GC (n=76),using immunohis to chemistry, as well as,the involvement of HIF pathway in GC cell migration and invasion in vitro. The correlation between HIF/ANXA1 and the prognosis wasalso evaluated.Results: HIF1αwas substantiallyupregulatedin GC tissues, compared to that of non-cancer. HIF1α was positively correlated withlymph node metastasis, but inversely correlated with survival in GC patients. HIF1αwas an independent prognostic factor in GC patients, and the combination of HIF1α and ANXA1 might serve asuseful prognostic markers in GCpatients.Furthermore,HIF-1αseems topromote GC migration and invasion through HIF1α/ANXA1/MMP-2 pathway. Conclusions: Our findings suggestthat HIF-1αmay serve as a promising prognostic biomarkerand therapeutic target for inhibiting GC metastasis.

Rhei Radix et Rhizoma is a kind of commonly used Chinese medicinal materials. Due to the overharvesting, the wild resource is endangering. Large market demand caused severely adulterant of commercial Rhei Radix et Rhizoma medicinal materials and decoction pieces. This manuscript reviewed the advances of the original species authentication in the industrial chain of Rhei Radix et Rhizoma during the latest decade, including characteristics and microscopic features, phytochemical analysis on anthraquinones, and molecular authentication based on DNA barcoding. Accordingly, an original species authentication route for the industrial chain of Rhei Radix et Rhizoma was summarized:(1)the identification of seeds and seedlings by DNA barcoding;(2) the selection of high variable sites based on the chloroplast genome;(3)biomonitoring of the Rhei Radix et Rhizoma medicinal materials and decoction pieces by two-dimensional DNA barcode;(4)traceability of Chinese patent medicines by third-generation sequencing. In conclusion, the combination of molecular identification and traditional identification methods provides a new idea for the identification of the original species of Rhei Radix et Rhizoma in the industrial chain and a essential guidance for the research of drug safety and efficacy of Rhei Radix et Rhizoma.

qRT-PCR was used for gene expression analysis. The subcellular localization of SmbHLH92 was detected byThese data indicate that

Bubali cornu (water buffalo horn) has been used as the substitute for Cornu rhinoceri asiatici (rhino horn) in clinical applications, and is the essential ingredient of Angong Niuhuang Wan. In recent years, there are a number of adulterants on the commercial herbal medicine markets. An efficient tool is required for species identification. In this study, 155 Bubali cornu samples have been taken from original animals and collected from commercial herbal medicine markets. 153 COI sequences have been successfully obtained from 155 samples through DNA extraction, PCR amplification, bidirectional sequencing and assembly. 93 COI sequences have been added to the DNA barcoding database of traditional Chinese animal medicine after validation using DNA barcoding GAP and tree-based methods. The species identification of the 62 commercial Bubali cornu medicines has been accomplished on the DNA barcoding system for identifying herbal medicine using the updated animal medicine database (www.tcmbarcode.cn). Except two samples failed to obtain COI sequences, 54.8% of the commercial Bubali cornu medicines were water buffalo horns and 29% were yak horns. Our results showed that yak horn was the major adulterant of Bubali cornu and the DNA barcoding method may accurately discriminate Bubali cornu and their adulterants. Therefore, we recommend that supervision on the herbal medicine markets should be strengthened with this new method to warren the effectiveness of herbal medicines.

To identify the commercial medicinal materials of Bombyx Batryticatus, two-dimensional DNA barcode was used to construct the "Internet Plus" identification system for Chinese medicine, which should benefit the cross-platform communication of DNA barcode information. Bombyx Batryticatus contained Bombyx mori Linnaeus and Beauveria bassiana (Bals.) Vuillant. Both COI and ITS sequences were obtained via PCR amplification for total genomic DNA extracted from raw materials using the animal genomic DNA kit, while only ITS but no COI sequences was obtained when using the plant genomic DNA kit. The ITS sequences obtained using the animal genomic DNA kit were consistent with those using plant genomic DNA kit. The medicinal materials yielded COI sequences and identified as B. mori. According to analysis of ITS sequences, the main species of the medicinal materials were identified as B. bassiana and few were identified as other fungi. NJ trees analysis based on ITS sequences suggests that it can be easily distinguished from other fungi. Our results showed that total genomic DNA of B. mori and B. bassiana was extracted simultaneously using the animal genomic DNA kit, which could effectively solve the problem in species identification of animal and fungi mixture materials. COI and ITS regions as DNA barcodes can stably and accurately identify Bombyx Batryticatus. The "Internet Plus" two-dimensional DNA barcode system will promote the standardization and normalization of Chinese medicinal materials market.

Traditional Chinese medicine (TCM) has contributad greatly to improving human health. However, the biological characteristics and molecular mechanisms of TCM in the treatment of human diseases remain largely unknown. Genomics plays an important role in modern medicine and biology. Here, we introduce genomics and other related omics to the study of herbs to propose a new discipline, Herbgenomics, that aims to uncover the genetic information and regulatory networks of herbs and to clarify their molecular mechanisms in the prevention and treatment of human diseases. Herbgenomics includes herbal structural genomics, functional genomics, transcriptomics, proteomics, metabonomics, epigenomics and metagenomics. Genomic information, together with transcriptomic, proteomic, and metabolomic data, can therefore be used to predict secondary metabolite biosynthetic pathways and their regulation, triggering a revolution in discovery-based research aimed at understanding the genetics and biology of herbs. Herbgenomics provides an effective platform to support chemical and biological analyses of complex herbal products that may contain more than one active component. Herbgenomics is now being applied to many areas of herb related biological research to help understand the quality of traditional medicines and for molecular herb identification through the establishment of an herbal gene bank. Moreover, functional genomics can contribute to model herb research platforms, geoherbal research, genomics-assisted herb breeding, and herbal synthetic biology, all of which are important for securing the future of medicinal plants and their active compounds. In addition, Herbgenomics will facilitate the elucidation of the targets and mechanism of herbs in disease treatment and provide support for personalized precise medicine.Herbgenomics will accelerate the application of cutting-edge technologies in herbal research and provide an unprecedented opportunity to revolutionize the use and acceptance of traditional herbal medicines.

A typical clinical case of taking Dictamni Cortex(Baixianpi) powder was analyzed to study liver damage caused by Dictamni Cortex. Liver damage was diagnosed according to the integrated evidence chain method recommended by the Guideline for Diagnosis and Treatment of Herb-Induced Liver Injury. By analyzing clinical history and biochemistry and imaging examinations, underlying diseases, such as viral hepatitis, autoimmune liver disease and alcoholic liver disease, were excluded. Through the investigation of medication history, we made it clear that the patient only took Dictamni Cortex powder during the period, and thus suspected that the liver injury was induced by Dictamni Cortex. Furthermore, the quality of the drug was tested, and the results showed it was consistent with the quality standard of Chinese Pharmacopoeia. DNA barcoding showed that the drug was 100% similar with Dictamnus dasycarpus. Moreover, exogenous harmful substances and chemical drug additions were tested, and the results showed that the content of heavy metal, pesticide residues and microbial toxin were consistent with the required standards, and no chemical drug additions were found in Agilent Fake TCM-Drugs database. In summary, we confirmed that the clinical case of drug-induced liver injury was induced by D. dasycarpus with the dose of 15 g•d⁻¹, which exceeded the prescribed amount of Chinese Pharmacopoeia. According to the Guideline for Diagnosis and Treatment of Herb-Induced Liver Injury, the case of drug-induced liver injury induced by D. dasycarpus was confirmed, which provided a direct and reliable evidence for the study of risk of liver injury induced by D. dasycarpus and its relevant preparations.

In order to authenticate the components of antler powder in the market, DNA barcoding technology coupled with cloning method were used. Cytochrome c oxidase subunit I (COI) sequences were obtained according to the DNA barcoding standard operation procedure (SOP). For antler powder with possible mixed components, the cloning method was used to get each COI sequence. 65 COI sequences were successfully obtained from commercial antler powders via sequencing PCR products. The results indicates that only 38% of these samples were derived from Cervus nippon Temminck or Cervus elaphus Linnaeus which is recorded in the 2010 edition of "Chinese Pharmacopoeia", while 62% of them were derived from other species. Rangifer tarandus Linnaeus was the most frequent species among the adulterants. Further analysis showed that some samples collected from different regions, companies and prices, contained adulterants. Analysis of 36 COI sequences obtained by the cloning method showed that C. elaphus and C. nippon were main components. In addition, some samples were marked clearly as antler powder on the label, however, C. elaphus or R. tarandus were their main components. In summary, DNA barcoding can accurately and efficiently distinguish the exact content in the commercial antler powder, which provides a new technique to ensure clinical safety and improve quality control of Chinese traditional medicine

Background: This study aims to determine the candidate markers that can be used as DNA barcode in the Lauraceae family. Material and Methods: Polymerase chain reaction amplification, sequencing efficiency, differential intra- and interspecific divergences, DNA barcoding gap, and identification efficiency were used to evaluate the four different DNA sequences of psbA-trnH, matK, rbcL, and ITS2. We tested the discrimination ability of psbA-trnH in 68 plant samples belonging to 42 species from 11 distinct genera and found that the rate of successful identification with the psbA-trnH was 82.4% at the species level. However, the correct identification of matK and rbcL were only 30.9% and 25.0%, respectively, using BLAST1. The PCR amplification efficiency of the ITS2 region was poor; thus, ITS2 was not included in subsequent experiments. To verify the capacity of the identification of psbA-trnH in more samples, 175 samples belonging to 117 species from the experimental data and from the GenBank database of the Lauraceae family were tested. Results: Using the BLAST1 method, the identification efficiency were 84.0% and 92.3% at the species and genus level, respectively. Conclusion: Therefore, psbA-trnH is confirmed as a useful marker for differentiating closely related species within Lauraceae.

In this paper, the chloroplast psbK-psbI intergenic spacers of 18 species of Dendrobium and their adulterants were amplified and sequenced, and then the sequence characteristics were analyzed. The sequence lengths of chloroplast psbK-psbI regions of Dendrobium ranged from 474 to 513 bp and the GC contents were 25.4%-27.6%. The variable sites were 71 while the informative sites were 46. The inter-specific genetic distances calculated by Kimura 2-parameter (K2P) of Dendrobium were 0.006 1-0.058 1, with an average of 0.028 4. The K2P genetic distances between Dendrobium species and Bulbophyllum odoratissimum were 0.093 2-0.120 4. The NJ tree showed that the Dendrobium species can be easily differentiated from each other and 6 samples of the inspected Dendrobium species were identified successfully through sequencing the psbK-psbI intergenic spacer. Therefore, the chloroplast psbK-psbI intergenic spacer can be used as a candidate marker to identify Dendrobium species and its adulterants.

RNA-Sequencing (RNA-Seq) is a newly-developed method in transcriptome research, it can afford more accurate transcription information and be more quickly by using Next-generation Sequencing (NGS) technology. RNA-Seq has been widely used in various biological fields. Genuine traditional Chinese medicines (TCM), with good quality and therapeutic effect, were always praised highly and used by famous physicians. The geo-herbalism formation of TCM is based on the product of the gene expression at specific space and time. So it has been a research hotspot to analyze the mechanism of biosynthesis through RNA-Seq in the study on the secondary metabolism of medicinal plant. This article mainly illustrates the RNA-Seq and its advantages, it also discusses the potential application in genuine TCM, and it can provide useful information for other researchers.

In order to identify Peucedani Radix, Peucedani Decursivi Radix and their adulterants, the internal transcribed spacer 2 (ITS2) regions of Peucedani Radix, Peucedani Decursivi Radix and their adulterants were amplified and bidirectionally sequenced based on the Principles for Molecular Identification of Traditional Chinese Materia Medica Using DNA Barcoding, which has been promulgated by Chinese Pharmacopoeia Commission. Sequences were analyzed and assembled by Codon Code Aligner V3. 7.1. The relevant data were analyzed by MEGA 5. 0. Species identification analyses were performed by using the nearest distance methods and neighbor-joining (NJ) methods. The result showed that the ITS2 sequence lengths of Peucedani Radix were 229-230 bp and the average intra-specific genetic distances were 0.005. The ITS2 sequence lengths of Peucedani Decursivi Radix were 227 bp and the sequences contained no variation site. The average inter-specific K2P genetic distance of Peucedani Radix, Peucedani Decursivi Radix and their adulterants species were 0.044 and 0.065 respectively. The minimum inter-specific divergence is larger than the maximum intra-specific divergence of Peucedani Decursivi Radix. The nearest distance methods and NJ trees results indicated that Peucedani Radix, Peucedani Decursivi Radix and their adulterants species could be identification clearly. The ITS2 regions can stably and accurately distinguish Peucedani Radix, Peucedani Decursivi Radix and their adulterants.

The medicinal fungi, which are of great importance in traditional medicine, are facing the problems of wild resources scarcity and low concentration of bioactive compounds. Velvet family and LaeA global regulator play a vital role in secondary metabolism and developmental programs, which are found in a wide variety of fungi ranging from Chytridiomycota to Basidiomycota. This review elaborates the structures and functions between Velvet family and LaeA protein. The Velvet family which shares the Velvet protein domain, including VeA (Velvet), VelB (Velvet like B), VosA (viability of spores A) and VelC (Velvet like C), acts on the regulation function is secondary metabolism and developmental programs such as asexual and sexual development. Furthermore, the function is affected by environmental factors such as light and temperature. LaeA protein which owns S-adenosylmethionine-dependent methyltransferase domain, coordinately regulates development and secondary metabolism by regulating and modifying the Velvet proteins. The regulation of LaeA is mediated by light receptor proteins. Therefore, clarifying the mechanism of Velvet and LaeA proteins in medicinal fungi will pave the way for nurturing medicinal fungi and improving production of bioactive compounds.

Codon usage bias is an important characteristic of genetic information transfer in organisms. Analysis of codon usage bias of different species is important for understanding the rules on genetic information transfer. The previous method for analysis of codon usage bias is mainly based on genomic data. However, this method is greatly limited, because the genome sequences of higher organisms are still not available up to now. In this study, we found that we could obtain the same optimal codons of Ganoderma lucidum (Curtis: Fr.) P. Karst based on its whole genomic data or large-scale transcriptomic data from its liquid-cultured hyphae, primordium and fruiting body, separately. This result indicated the feasibility to understand the codon usage bias based on the large-scale transcriptomic data. By calculating the proportion of rare codons of Escherichia coli and Saccharomyces cerevisiae in 26 terpene synthases (TS) of G. lucidum, we found that the rare codons of S. cerevisiae have a higher proportion in TS genes, while the rare codons of E. coli have relatively lower, suggesting that the TS genes of G. lucidum are possibly more difficult to be expressed in S. cerevisiae than in E. coli. Chemical synthesis of TS genes according to the yeast optimal codons will be an effective way to solve the problem on the mismatch of gene codon bias between the foreign genes and the host strain.

In order to construct an integrated DNA barcoding database for identifying Chinese animal medicine, the authors and their cooperators have completed a lot of researches for identifying Chinese animal medicines using DNA barcoding technology. Sequences from GenBank have been analyzed simultaneously. Three different methods, BLAST, barcoding gap and Tree building, have been used to confirm the reliabilities of barcode records in the database. The integrated DNA barcoding database for identifying Chinese animal medicine has been constructed using three different parts: specimen, sequence and literature information. This database contained about 800 animal medicines and the adulterants and closely related species. Unknown specimens can be identified by pasting their sequence record into the window on the ID page of species identification system for traditional Chinese medicine (www. tcmbarcode. cn). The integrated DNA barcoding database for identifying Chinese animal medicine is significantly important for animal species identification, rare and endangered species conservation and sustainable utilization of animal resources.

Fifty-eight samples belonging to 7 species of Arisaematis Rhizoma and its adulterants were collected. The ITS2 locus was employed as a DNA barcode and amplified, sequenced and assembled for all of the collected samples. Then, ITS2 sequences have been annotated using HMM-based method. The intra- and inter-specific variations were calculated and NJ tree was constructed using MEGA 6.0 software. The results showed that inter-specific K2P distances were significantly larger than intra-specific distances for all of the three origin species of Arisaematis Rhizoma. Furthermore, three origin species, Arisaema amurense, A. erubescens and A. heterophyllum, can be respectively formed to be a single branch with high bootstrap values. It is concluded that ITS2 can be used to correctly identify Arisaematis Rhizoma from its adulterants and the application of ITS2 in the identification of traditional Chinese medicine has an important prospective.

In order to identify Aucklandiae Radix, Vladimiriae Radix, Inulae Radix, Aristolochiae Radix and Kadsurae Radix using ITS2 barcodes, genomic DNA from sixty samples was extracted and the ITS2 (internal transcribed spacer) regions were amplified and sequenced. The genetic distances were computed using MEGA 5.0 in accordance with the kimura 2-parameter (K2P) model and the neighbor-joining (NJ) phylogenetic tree was constructed. The results indicated that for Aucklandiae Radix (Aucklandia lappa), Vladimiriae Radix (Vladimiria souliei and V. souliei var. cinerea), Inulae Radix (Inula helenium), Aristolochiae Radix (Aristolochia debilis) and Kadsurae Radix (Kadsura longipedunculata), the intra-specific variation was smaller than inter-specific one. There are 162 variable sites among 272 bp after alignment of all ITS2 sequence haplotypes. For each species, the intra-specific genetic distances were also smaller than inter-specific one. Furthermore, the NJ tree strongly supported that Aucklandiae Radix, Vladimiriae Radix, Inulae Radix, Aristolochiae Radix and Kadsurae Radix can be differentiated. At the same time, V. souliei (Dolomiaea souliei) and V. souliei var. cinerea( D. souliei var. cinerea) belonging to Vladimiriae Radix were clearly identified. In conclusion, ITS2 barcode could be used to identify Aucklandiae Radix, Vladimiriae Radix, Inulae Radix, Aristolochiae Radix and Kadsurae Radix. Our study may provide a scientific foundation for clinical safe use of the traditional Chinese medicines.

In this study, the COI barcode was used to identify the Scolopendra medicinal materials and its adulterants in order to provide a new method for the identification of Scolopendra. Genomic DNA was extracted from the experimental samples. The COI sequences were amplified and sequenced bi-directionally. Sequence alignment and NJ tree construction was carried out by MEGA6.0 software. The results showed that the COI sequences can be obtained from all experimental samples. The average inter-specific K2P distance of Scolopendra was 0.222 and the minimum inter-specific distance was 0.190. All the Scolopendra subspinipes mutilans medicinal samples clustered into a clade in the NJ tree and can be distinguished from its adulterants. In a conclusion, COI can be used to correctly identify Scolopendra medicinal materials, and it will be a potential DNA barcode for identifying other animal medicinal materials.

The ITS2 barcode was used to accurately identify Albiziae Cortex, Albiziae Flos and their adulterants in this study. A total of46 samples from Albiziae Cortex, Albiziae Flos and their adulterants were collected. The ITS2 regions were amplified and sequenced. Sequences were assembled using the CodonCode Aligner. The genetic distances of ITS2 region were calculated using MEGA 5.0. BLAST1, nearest distance and phylogenetic tree (NJ-tree) methods were used to assess the identification efficiency of the ITS2 barcode. The results revealed that the intraspecific genetic distances of Albizia julibrissin were lower than the interspecific genetic distances between A. julibrissin and its adulterants. The identification efficiency of ITS2 barcode using BLAST1 was 100%. The NJ-tree showed that A. julibrissin and their adulterants can be easily differentiated according to their monophyly. The ITS2 barcode is suitable to be as a barcode to identify Albiziae Cortex, Albiziae Flos and their adulterants.

Transcription factor is one of the key factors in the regulation of gene expression at the transcriptional level. It plays an important role in plant growth, active components biosynthesis and response to environmental change. This paper summarized the structure and classification of bHLH transcription factors and elaborated the research progress of bHLH transcription factors which regulate the active components in plants, such as flavonoids, alkaloids, and terpenoids. In addition, the possibility of increasing the concentration of active substances by bHLH in medicinal plants was assessed. The paper emphasized great significance of model plants and multidisciplinary research fields including modern genomics, transcriptomics, metabolomics and bioinformatics, providing the contribution to improve the discovery and function characterization of bHLH transcription factors. Accelerating the research in the mechanism of bHLH transcription factors on the regulation of active components biosynthesis will promote the development of breeding and variety improvement of Chinese medicinal materials, also ease the pressure of resources exhaustion of traditional Chinese medicine home and abroad.

In order to construct an integrated DNA barcoding database for identifying Chinese animal medicine, the authors and their cooperators have completed a lot of researches for identifying Chinese animal medicines using DNA barcoding technology. Sequences from GenBank have been analyzed simultaneously. Three different methods, BLAST, barcoding gap and Tree building, have been used to confirm the reliabilities of barcode records in the database. The integrated DNA barcoding database for identifying Chinese animal medicine has been constructed using three different parts: specimen, sequence and literature information. This database contained about 800 animal medicines and the adulterants and closely related species. Unknown specimens can be identified by pasting their sequence record into the window on the ID page of species identification system for traditional Chinese medicine (www. tcmbarcode. cn). The integrated DNA barcoding database for identifying Chinese animal medicine is significantly important for animal species identification, rare and endangered species conservation and sustainable utilization of animal resources.

A Ti transformed cell culture of S. miltiorrhiza demonstrated the ability to produce rosmarinic acid (RA) and lithospermic acid B (LAB) as determined by HPLC. A time course of biomass accumulation, phenolic compound formation, medium pH and conductivity as well as sugar, nitrate, ammonium and phosphate uptake was examined both in flask and in bioreactor cultures using hormone free B5 medium containing 2% sucrose. In flasks, the stationary phase of growth was reached after about 16 days of cultivation; the maximum biomass concentration was about 10.5 g l −1 dry weight. The maximum production of RA (4.59% of dry cell weight or 481.5 mg l −1 ) appeared on the 20th day of cultivation, at which time the production of LAB was 0.81% of dry weight or 85.0 mg l −1 . A linear relationship with an intercept between the dry cell weight increase and the medium conductivity decrease was observed which may be used for monitoring cell growth rate. Initia1 medium pH over a wide range (3.4–8.5) had little effect on cell growth but had significant influences on rosmarinic acid formation, although the trend was irregular. In bioreactor cultivation, the maximum dry cell biomass accumulation obtained was 7.52 g l −1 on the 15th day of cultivation. The lower performance of cell growth in the bioreactor was due to the poor mixing of the culture.

Transformed cell cultures of Salvia miltiorrhiza were established by infecting sterile plantlets with Agrobacterium tumefaciens strain C58. The transformed cells in suspension formed macroscopic clumps of cell aggregates up to 2-3 cm in size rather than homogeneous cell suspensions. These transformed cells grew well in hormone-free media. It was found that the B5 mediums supported the best growth while the 6,7-V medium promoted tanshinone production in the transformed cell suspension cultures. The effect of initial sucrose concentration on cell growth was also studied. The best growth was observed when cells were cultivated in the B5 medium containing 30 g l-1 sucrose. Although low levels of tanshinones were produced in fast growing cell cultures, there existed a rapid increase in tanshinone production when the cell aggregates were transferred to the fresh yeast-extract-containing medium. By this two-stage culture method, about 22 mg tanshinones were produced in 1 liter of medium. Green cell aggregates were formed when cells were cultured under illumination. Light was found to have an inhibitory effect on tanshinone biosynthesis. A sensitive high-performance liquid chromatographic method was developed for the measurement of tanshinones. Cryptotanshinone, tanshinone I and tanshinone IIA were identified from the transformed cultures.

Research on medicinal model organism is one of the core technologies to promote the modernization of traditional Chinese medicine (TCM). The research progress of Salvia miltiorrhiza as medicinal model plant is summarized in this paper. The genome of S. miltiorrhiza is small and its life cycle is short, as well as this plant can be stably genetically transformed. Because S. miltiorrhiza possesses the important medicinal and economic values, recently the transcriptome and genome of S. miltiorrhiza have been significantly recovered. The research prospect of S. miltiorrhiza as medicinal model plant in TCM was discussed, including biosynthesis of active components and their genetic regulation, relationship between quality of TCM and ecological environments, and selective breeding of good quality lines. Furthermore, as medicinal model plant, the construction of mutant library for S. miltiorrhiza, the genome map with high quality, and the functional genome should be investigated. Accompanying modern investigation of life sciences, the platform for medicinal model plant, S. miltiorrhiza, will be promoted to be established. It is important to develop the ethnopharmacology and new drugs around the world.

Quality variation and ecotype classification of Chinese herbal medicine are important scientific problems in Daodi herbal medicine research. The diversity of natural environmental conditions has led to form unique multi-Daodi, multi-product areas that produce particular Chinese herbal medicine. China is one of three big American ginseng (Panax quinquefolium L.) producing areas worldwide, with over 300 years of application and 40 years of cultivation history. Long-term production practice has led to the formation of three big advocate produce areas in China: Northeast province, Beijing and Shandong. P. quinquefolium L. grown under certain environmental conditions will develop long-term adaptations that will lead to more stable strains (different ecotypes). P. quinquefolium L., can vary greatly in quality; however, the ecological mechanisms causing this variation are still unclear. Root samples were collected from four-year-old cultivated P. quinquefolium L. plants in the three major genuine (Daodi) American ginseng-producing areas of Northeast province, Beijing and Shandong province, China. Ultra-performance liquid chromatography was used to analyze the contents of eight ginsenosides (Rg1, Re, Rb1, Rb2, Rb3, Rc, Rd, Rg2). Data for nine ecological factors, including temperature, moisture and sunlight, were obtained from the ecological database of Geographic Information System for Traditional Chinese Medicine. Soil samples from the sampling sites were collected. Effective boron and iron, available nitrogen and potassium, as well as other trace elements and soil nutrients, were determined by conventional soil physicochemical property assay methods. Analytical methods of biostatistics and numerical taxonomy were used to divide ecotypes of the three main Panax quinquefolium L. producing areas in China based on ginsenoside content, climate, soil and other ecological factors. To our knowledge, this is the first time that ecological division of P. quinquefolium L. producing areas in China has ever been conducted. The results show that there are two chemoecotypes of P. quinquefolium L. in China: ginsenoside Rb1-Re from outside Shanhaiguan, and ginsenoside Rg2-Rd from inside Shanhaiguan. Similarly, there are two types of climatic characteristics: inside Shanhaiguan (Beijing, Shandong) and outside Shanhaiguan (Northeast). This suggests that the formation and differentiation of chemoecotypes of P. quinquefolium L. is closely related to variability of the climatic and geographical environment. Additionally, ecological variation of the three main producing areas, characteristics of two climatic ecotypes, and soil characteristics are also discussed and summarized. These results provide experimental scientific evidence of the quality variation and ecological adaptation of P. quinquefolium L. from different producing areas. They also deepen our understanding of the biological nature of Daodi P. quinquefolium L. formation, and offer novel research models for other multi-origin, multi-Daodi Chinese herbal medicines ecotypes. In addition, the results demonstrate the critical need for improving quality, appropriate ecological regionalization and promoting industrialized development of P. quinquefolium L.

The authors reviewed the new technologies used for Panax genus research, including molecular identification technologies (especially for DNA barcoding), modern biotechnologies (e. g. the first generation and second generation sequencing technologies), and gene cloning and identification in this paper. These technologies have been successfully applied to species identification, transcriptome analysis, secondary metabolite biosynthetic pathway and the key enzyme function identification, indicating that the application of modern biotechnologies provide guarantee for the molecular identification of Panax genus. The application of modern biotechnologies also reveals the genetic information of transcriptome and functional genomics, and promotes the design of Panax plants genomic map. In summary, the application of the new technologies lay the foundation for clarifying the molecular mechanisms of ginsenoside biosynthesis and enforcing the in vitro synthesis of important natural products and new drugs in future.

Cytochrome P450 (CYP450) is a key element in the Ganoderma triterpenoid biosynthetic pathway. The catalytic reaction process for CYP450 requires NADPH / NADH for electron transfer. After searching the genome dataset of Ganoderma lucidum, the unique sequence encoding CYP450 and NADPH were discovered, separately. The open reading frames of GLCYP450 and GLNADPH were cloned separately using RT-PCR strategy from G lucidum. The appropriate restriction enzyme cutting sites were introduced at the 5' and 3' ends of gene sequence. The genes of GLCYP450 and GLNADPH were recombined into the yeast expression vector pESC-URA, leading to the formation of the yeast expression plasmid pESC-GLNADPH-GLCYP450. This study provides a foundation for researching Ganoderma triterpene biosynthesis using the approach of synthetic biology.

Since the research of molecular identification of Chinese Materia Medica (CMM) using DNA barcode is rapidly developing and popularizing, the principle of this method is approved to be listed in the Supplement of the Pharmacopoeia of the People's Republic of China. Based on the study on comprehensive samples, the DNA barcoding systems have been established to identify CMM, i.e. ITS2 as a core barcode and psbA-trnH as a complementary locus for identification of planta medica, and COI as a core barcode and ITS2 as a complementary locus for identification of animal medica. This article introduced the principle of molecular identification of CMM using DNA barcoding and its drafting instructions. Furthermore, its application perspective was discussed.

DNA barcoding is a rapidly developing frontier technology in the world and will be useful in promoting the quality control and standardization of traditional Chinese medicine. Until now, many studies concerning DNA barcoding have focused on leaf samples but rarely on Chinese herbal medicine. There are three issues involved in DNA barcoding for traditional Chinese medicinal materials: (1) the extraction methods for total DNA of the rhizomes of the medicinal materials; (2) intra-specific variation among samples from different places of origin; (3) accuracy and stability of this method. In this study, Gentianae Macrophyllae Radix was used to verify the stability and accuracy of DNA barcoding technology. Five regions (ITS2, psbA-trnH, matK, rbcL, and ITS) were tested for their ability to identify 86 samples of Gentianae Macrophyllae Radix and their adulterants. After improving the DNA extraction method, genomic DNA from all samples was successfully obtained. To evaluate each barcode's utility for species authentication, PCR amplification efficiency, genetic divergence, and species authentication were assessed. Among all tested regions only ITS2 locus showed 100% of PCR amplification and identification efficiencies. Based on the established method, we successfully identified two samples of Gentianae Macrophyllae Radix bought in pharmacy to the original species.

Traditional Chinese medicine (TCM) genomics and TCM synthetic biology are two hot fields in the TCM modernization. TCM genomics, including transcriptomics, structural genomics, genomic markers and functional genomics, aims to elucidate the biosynthetic pathways of TCM bioactive compounds and mine the related genes encoding enzymes involved in these pathways by analyzing genetic information on the original species of TCM, thus promoting the development of TCM synthetic biology, genome-assisted molecular identification and molecular breeding, and elucidation of the genetic mechanism underlying "Daodi". Genomics and related research provide us much deeper understanding of life process and improve our ability to create new life or modify the present organisms. Based on TCM genomics, TCM synthetic biology sets up a series of procedures to realize the production of TCM pharmacological active compounds in microorganism, including screening and modification of parts and devices, establishment of standardized part and device libraries, and reconstruction and modification of the biosynthetic pathway of TCM pharmacological active compounds in microorganism. TCM synthetic biology will provide a new resource of TCM pharmacological active compounds for the pharmacological study and researchdevelopment of new drugs, thus enhancing the core competitiveness of our pharmaceutical industry in the international markets.

In this study, Notopterygii Rhizoma et Radix was used to verify the stability and accuracy of DNA barcodes in identification of Chinese materia medica for the first time. All genomic DNAs from thirty one samples were extracted. The ITS (internal transcribed spacer) regions were amplified and sequenced bi-directionally. Obtained sequences were assembled using the CodonCode Aligner. And the sequences of the ITS regions were aligned through Clustal-W and the genetic distances were computed using MEGA 5.0 in accordance with the kimura 2-parameter (K2P) model. The neighbor-joining (NJ) phylogenetic trees were constructed. The ITS2 regions were obtained by using the hidden Markov model (HMM)-based annotation methods from the ITS sequences. Results indicated that the lengths of ITS regions of Notopterygii Rhizoma et Radix were 603-604 bp, while the lengths of ITS2 regions were 228 bp. The haplotypes of ITS/ITS2 regions of Notopterygii Rhizoma et Radix were the same as those of the original plant leaves. The intra-specific genetic distances were smaller than inter-specific ones in ITS/ITS2 regions of Notopterygium incisum and N. franchetii. The NJ trees showed that N. incisum, N. franchetii and its adulterants can be easily differentiated according to their monophyly. Therefore, ITS/ITS2 regions as DNA barcodes can stably and accurately distinguish Notopterygii Rhizoma et Radix from its adulterants and could provide a new technique to ensure clinical safety in utilization of traditional Chinese medicines.

The dried succulent stems of Cistanche (Cistanche deserticola Y. C. Ma and Cistanche tubulosa Wight.) are one of the most widely used components of traditional Chinese medicines. However, it is often confused and substituted with the roots of Orobanche pycnostachya, Boschniakia rossica (Cham.Schltdl.) Standl., Cistanche sinensis Beck, and Cistanche salsa (C. A. Mey.) Beck. In this study, we identified psbA-trnH regions from species and tested their suitable for the identification of the above mentioned taxa. The psbA-trnH sequences showed considerable variations between species and thus were revealed as a promising candidate for barcoding of Cistanche species. Additionally, the average genetic distance of psbA-trnH ranging from 0.077% to 0.743%. In contrast, the intra-specific variation among Cistanche species was found to be significantly different from those of other species, with percentages of variation studied ranged from 0% to 0.007%. The sequence difference between the psbA-trnH sequences of Cistanche species and Orobanche pycnostachya ranged from 0.979% to 1.149%. The distance between the Cistanche species and Boschniakia rossica ranged from 1.066% to 1.224%. Our results suggest that the psbA-trnH intergenic spacer region represent a barcode that can be used to identify Cistanche species and other morphologically undistinguishable species.

Medicinal pteridophytes are an important group used in traditional Chinese medicine; however, there is no simple and universal way to differentiate various species of this group by morphological traits. A novel technology termed "DNA barcoding" could discriminate species by a standard DNA sequence with universal primers and sufficient variation. To determine whether DNA barcoding would be effective for differentiating pteridophyte species, we first analyzed five DNA sequence markers (psbA-trnH intergenic region, rbcL, rpoB, rpoC1, and matK) using six chloroplast genomic sequences from GeneBank and found psbA-trnH intergenic region the best candidate for availability of universal primers. Next, we amplified the psbA-trnH region from 79 samples of medicinal pteridophyte plants. These samples represented 51 species from 24 families, including all the authentic pteridophyte species listed in the Chinese pharmacopoeia (2005 version) and some commonly used adulterants. We found that the sequence of the psbA-trnH intergenic region can be determined with both high polymerase chain reaction (PCR) amplification efficiency (94.1%) and high direct sequencing success rate (81.3%). Combined with GeneBank data (54 species cross 12 pteridophyte families), species discriminative power analysis showed that 90.2% of species could be separated/identified successfully by the TaxonGap method in conjunction with the Basic Local Alignment Search Tool 1 (BLAST1) method. The TaxonGap method results further showed that, for 37 out of 39 separable species with at least two samples each, between-species variation was higher than the relevant within-species variation. Thus, the psbA-trnH intergenic region is a suitable DNA marker for species identification in medicinal pteridophytes.

DNA barcoding is a rapidly developing frontier technology that is gaining worldwide attention. Here, seven regions (psbA-trnH, matK, ycf5, rpoC1, rbcL, ITS2, and ITS) with potential for use as DNA barcodes were tested for their ability to identify 300 samples of 192 species from 72 genera of the family Rutaceae. To evaluate each barcode’s utility for species authentication, PCR amplification efficiency, genetic divergence, and barcoding gaps were assessed. We found that the ITS2 region exhibited the highest inter-specific divergence, and that this was significantly higher than the intra-specific variation in the “DNA barcoding gap” assessment and Wilcoxon two-sample tests. The ITS2 locus had the highest identification efficiency among all tested regions. In a previous study, we found that ITS2 was able to discriminate a wide range of plant taxa, and here we confirmed that ITS2 was also able to discriminate a number of closely related species. Therefore, we propose that ITS2 is a promising candidate barcode for plant species identification.

To investigate the profile of gene expression in American ginseng (Panax quinquefolium L.) and discover its functional genes, for the first time, expressed sequence tags (EST) library of four-year-old American ginseng roots has been established. According to BLAST and Gene Ontology analysis, eleven genes, encoding cytochrome P450, glucosyltransferase, farnesyltransferase and cyclase family protein, are found to be associated with ginsenosides biosynthesis. Six other genes are obtained encoding auxin-regulated protein, auxin response factor 4 and auxin-repressed protein in the roots of American ginseng. In addition, thirteen expressed transcripts are stress-connected proteins and twelve expressed other transcripts are closely related to plant defense in four-year-old American ginseng roots. Furthermore, 62 genes no hit in BLAST and in Interproscan may be new genes. These results indicate EST is an useful tool for research on functional genomics of P. quinquefolium and it can be applied to the molecular modification of the ginsenosides biosynthetic pathway ultimately for improving the quality of American ginseng germplasm.