Assembly and comparative analysis of the complete mitochondrial genome of Pinellia ternata.

Pinellia ternata is an important natural medicinal herb in China. However, it is susceptible to withering when exposed to high temperatures during growth, which limits its tuber production. Mitochondria usually function in stress response. The P . ternata mitochondrial (mt) genome has yet to be explored. Therefore, we integrated PacBio and Illumina sequencing reads to assemble and annotate the mt genome of P . ternata . The circular mt genome of P . ternata is 876 608bp in length and contains 38 protein-coding genes (PCGs), 20 tRNA genes and three rRNA genes. Codon usage, sequence repeats, RNA editing and gene migration from chloroplast (cp) to mt were also examined. Phylogenetic analysis based on the mt genomes of P . ternata and 36 other taxa revealed the taxonomic and evolutionary status of P . ternata . Furthermore, we investigated the mt genome size and GC content by comparing P . ternata with the other 35 species. An evaluation of non-synonymous substitutions and synonymous substitutions indicated that most PCGs in the mt genome underwent negative selection. Our results provide comprehensive information on the P . ternata mt genome, which may facilitate future research on the high-temperature response of P . ternata and provide new molecular insights on the Araceae family.

Identification of Andrographis Herba and its common products using mini-barcode.

Andrographis Herba, the aerial part of Andrographis paniculata (Burm. f.) Wall. ex Nees (Acanthaceae), has a wide geographic distribution and has been used for the treatment of fever, cold, inflammation, and other infectious diseases. In markets, sellers and buyers commonly inadvertently confuse with related species. In addition, most Chinese medicinal herbs are subjected to traditional processing procedures, such as steaming and boiling, before they are sold at dispensaries; therefore, it is very difficult to identify Andrographis Herba when it is processed into Chinese medicines. The identification of species and processed medicinal materials is a growing issue in the marketplace. However, conventional methods of identification have limitations, while DNA barcoding has received considerable attention as a new potential means to identify species and processed medicinal materials. In this study, 17 standard reference materials of A. paniculata, 2 standard decoctions, 27 commercial products and two adulterants were collected. Based on the ITS2 sequence, it could successfully identify A. paniculata and adulterants. Moreover, a nucleotide signature consisting of 71 bp was designed, this sequence is highly conserved and specific within A. paniculata while divergent among other species. Then, we used these new primers to amplify the nucleotide signature region from processed materials. In conclusion, the DNA barcoding method developed in the present study for authenticating A. paniculata is rapid and cost-effective. It can be used in the future to guarantee the quality of Andrographis Herba of each regulatory link for clinical use.

Analysis of DNA methylation in potato tuber in response to light exposure during storage.

Exposure to light induces tuber greening and the accumulation of the toxic alkaloid Solanine in potato (Solanum tuberosum L) during storage greatly reduce tuber value. While the mechanism of this greening process remains unclear, it is well understood that DNA methylation plays an important role in regulating gene expression in response to environmental conditions. In this study, methylation-sensitive amplified polymorphism was used to assess the effect of light exposure on DNA methylation during storage of potato tubers. Light-induced genome-wide DNA demethylation and the rate of DNA methylation decreased with long storage times. Following, the sequencing of 14 differentially amplified fragments and analysis using the Basic Local Alignment Search Tool, eight genomic sequences and six annotated fragment sequences were identified. The latter included ADP glucose pyrophosphorylase 1/2, chlorophyllide a oxygenase 1 (CAO1), receptor-like protein kinase HAIKU2, and repressor of GA4, all of which are involved in starch biosynthesis, chlorophyll synthesis, endosperm development, and gibberellic acid signaling, respectively. Demethylation was observed in the CpG island (-273 to -166 bp) of the CAO1 promoter in response to light, which further confirmed that the variations in genome methylation are dependent upon the light exposure and suggests a direct role for DNA methylation. Our results provide an epigenetic perspective for further exploring the mechanism of light-induced tuber greening.

[Research progress on mechanism of phytohormones in regulating flavonoid metabolism].

Phytohormones play an important role at all stages of plant growth, influencing plant growth and development and regulating plant secondary metabolism, such as the synthesis of flavone, flavonol, anthocyanin, and other flavonoids. Flavonoids, a group of important secondary metabolites ubiquitous in plants, have antioxidative, anti-microbial, and anti-inflammatory activities and thus have a wide range of potential applications in Chinese medicine and food nutrition. With the development of biotechnology, phytohormones' regulation on flavonoids has become a research focus in recent years. This study reviewed the research progress on the mechanism of common phytohormones, such as abscisic acid, gibberellin, methyl jasmonate, and salicylic acid, in regulating flavonoid metabolism, and discussed the molecular mechanism of the synthesis and accumulation of flavonoids, aiming at clarifying the key role of phytohormones in modulating flavonoid metabolism. The result is of guiding significance for improving the content of flavonoids in plants through rational use of phytohormones and of reference value for exploring the mechanism of hormones in regulating flavonoid metabolism.

UHPLC-MS-based metabolomic approach for the quality evaluation of Pinellia ternata tubers grown in shaded environments.

Pinellia ternata is a native herb in China, and its tuber is widely-used in traditional Chinese medicines. It has been identified that the shading treatment promotes tuber production during cultivation. However, the tuber quality in shaded environments is unknown, which limits the scientific cultivation of P. ternata. In this study, a metabolomics approach based on UHPLC-MS was applied to assess the metabolic components of P. ternata in response to shading. Diverse metabolites were profiled using the metabolomics approach. Then, datasets of P. ternata cultivated in natural light (control) and shaded environments were subjected to multivariate analyses. Two P. ternata tuber products were well separated by the PCA. In total, four P. ternata alkaloids with contents that were not altered by the shaded environment were detected. Metabolomic analyses further identified several organic acids [mevalonic acid, 12,13-dihydroxy-9Z-octadecenoic acid (12, 13-DiHOME), urocanic acid, and γ-aminobutyric acid] that were largely enriched in the shaded environment, which likely contributed to tuber quality and growth. This study determined that shading probably improves the quality of P. ternata tubers and laid a foundation for exploring the regulatory mechanism of the shade response in P. ternata.

[Regulation of ß-mercuryl alcohol metabolic flow in Saccharomyces cerevisiae cells].

In this study, citrate synthase gene(CIT2), and malate synthase gene(MLS1) were successfully knocked out in ß-amyrin-producing yeast cells by using CRISPR/CAS9. The promoter of phosphoglucose isomerase gene(PGI1) was replaced by that of cytochrome c oxidase subunit Ⅶa(Cox9)to weaken its expression, aiming to channel more carbon flux into the NADPH-producing pathway. The fermentation results showed that CIT2 deletion had no effect on the ß-amyrin production. Compared with the control strain, the production of ß-amyrin was increased by 1.85 times after deleting MLS1, reaching into 3.3 mg·L~(-1). By replacing the promoter of PGI1, the ß-amyrin yield was 3.75 times higher than that of the control strain, reaching up to 6.7 mg·L~(-1). This study successfully knocked out the CITT2 and MLS1 genes and weakened the PGI1 gene by using CRISPR/CAS9, which directly influenced the production of ß-amyrin and provided some reference for the the metabolic engineering of triterpernoid producing strain.

Liquid chromatography-mass spectrometry-based metabolomics analysis of flavonoids and anthraquinones in Fagopyrum tataricum L. Gaertn. (tartary buckwheat) seeds to trace morphological variations.

Polyphenols (flavonoids and anthraquinones) are one of the most important phytochemicals in Fagopyrum tataricum L. Gaertn. (tartary buckwheat). However, the relationship between the polyphenols of tartary buckwheat seeds and their morphological variations is unclear. We developed a liquid chromatography-mass spectrometry-based targeted metabolomics method to study the chemical profiles of 60 flavonoids and 11 anthraquinones in 40 seed cultivars (groats and hulls). Both flavonoids and anthraquinones were related to variations in seed color; the fold change from yellowish-brown to black seeds was 1.24-1.55 in groats and 0.26-0.76 in hulls. Only flavonoids contributed to significant differences in seed shape; the fold change from long to short seeds was 1.29-1.78 in groats and 1.39-1.44 in hulls. Some differential metabolites were identified at higher concentrations in hulls than in groats. This study provides new insights into differences in polyphenols among tartary buckwheat seeds with different color and shape.

[Variation analysis of genomic methylation induced by high temperature stress in Pinellia ternata].

Pinellia ternata belongs to the Araceae family and is a medicinal herb. The tuber is the medicinal organ with antitussive, antiemetic and anti-tumor activities. It is easy to encounter high temperature environment during the growth periods, leading to decrease of tuber production. At present, the mechanism of response to high temperature stress in P. ternata is still unknown. DNA methylation plays a vital role in plant protection against adversity stress as a way of epigenetic regulation. In this study, P. ternata was used as material for treatment of high temperature stress at 0 h, 6 h and 80 h, and methylation sensitive amplification polymorphism(MSAP) analysis was conducted on the changes of DNA methylation in its genome. The results showed that 20 pairs of MSAP primers were selected from 100 MSAP primers with multiple clear and uniform bands, and 353, 355 and 342 loci were amplified from materials of P. ternata treated in the high temperature stress 0 h, 6 h and 80 h, respectively. Cytosine methylation levels of CCGG context in the above materials were characterized as 60.91%, 44.79% and 44.74%, respectively. And the full methylation ratios were 16.71%, 22.25% and 29.24, respectively. It demonstrated that high temperature stress significantly induced the down-regulation of DNA methylation level and up-regulation of the full methylation rate in P. ternata genome. This study provides a preliminary theoretical reference for analyzing the mechanism of P. ternata responding to high temperature stress from the epigenetic perspective.

[Analysis of shading on DNA methylation by MSAP in Pinellia ternata].

Pinellia ternata is a medicinal herb of Araceae, and its tubers are used as medicines. It is a common Chinese herbal medicine in China and has a large market demand. When exposing to strong light intensity and high temperature during the growth process, P. ternata withers in a phenomenon known as "sprout tumble", which largely limits tuber production. Shade can effectively delay sprout tumble formation and increase its yield, however the relevant regulation mechanism is unclear. DNA methylation, as a self-modifying response to environmental changes, is often involved in the regulation of plant growth and development. In this study, P. ternata grown under natural light and 90% shading were selected as the control group and the experimental group for genomic DNA methylation analysis by using methylate sensitive amplification polymorphism(MSAP). The results showed that a total of 617 loci were detected with 20 pairs of primers, of which 311 were in the natural light group and 306 in the shading group. The methylation sites in the light and shading groups accounted for 58.2% and 71.57%, respectively, and the methylation ratios in the methylation sites were 27.65% and 29.41%, respectively, indicating that shading significantly induced the genome DNA methylation of P. ternata. Compared to the natural light group, shading promoted 32.51% of the genes methylation, while inducing 16.25% gene demethylation. This study reveals the DNA methylation variation of P. ternata under shading conditions, which lays a preliminary theoretical foundation for further analysis of the mechanism of shading regulation of P. ternata growth from epigenetic level.

Inducing Hairy Roots by Agrobacterium rhizogenes-Mediated Transformation in Tartary Buckwheat (Fagopyrum tataricum).

Tartary buckwheat (TB) [Fagopyrum tataricum (L.) Gaertn] possesses various biological and pharmacological activities because it contains abundant secondary metabolites such as flavonoids, especially rutin. Agrobacterium rhizogenes have been gradually used worldwide to induce hairy roots in medicinal plants to investigate gene functions and increase the yield of secondary metabolites. In this study, we have described a detailed method to generate A. rhizogenes-mediated hairy roots in TB. Cotyledons and hypocotyledonary axis at 7-10 days were selected as explants and infected with A. rhizogenes carrying a binary vector, which induced adventitious hairy roots that appeared after 1 week. The generated hairy root transformation was identified based on morphology, resistance selection (kanamycin), and reporter gene expression (green fluorescent protein). Subsequently, the transformed hairy roots were self-propagated as required. Meanwhile, a myeloblastosis (MYB) transcription factor, FtMYB116, was transformed into the TB genome using the A. rhizogenes-mediated hairy roots to verify the role of FtMYB116 in synthesizing flavonoids. The results showed that the expression of flavonoid-related genes and the yield of flavonoid compounds (rutin and quercetin) were significantly (p < 0.01) promoted by FtMYB116, indicating that A. rhizogenes-mediated hairy roots can be used as an effective alternative tool to investigate gene functions and the production of secondary metabolites. The detailed step-by-step protocol described in this study for generating hairy roots can be adopted for any genetic transformation or other medicinal plants after adjustment.

Systematic review and meta-analyses of lead (Pb) concentrations in environmental media (soil, dust, water, food, and air) reported in the United States from 1996 to 2016.

Environmental lead (Pb) contamination is a persistent public health issue that prominently impacts communities across the United States. Multimedia Pb exposure assessments are utilized to provide a holistic evaluation of Pb exposure and inform the development of programs and regulations that are protective of human health. To conduct multimedia exposure assessments, robust, media-specific environmental Pb concentration data are necessary. To support this effort, systematic review and meta-analysis methods were used to conduct a comprehensive synthesis of research measuring Pb in multiple environmental media (soil, dust, water, food, and air) over a 20-year period within the United States. The breadth of the resulting database allowed for the evaluation of sample characteristics that can serve as indicators of environmental Pb contamination. Random effects models run on literature and national survey datasets generated overall mean estimates of Pb concentrations that can be used for multimedia Pb exposure modeling for general and high-exposure-risk populations. Results from our study highlighted several important trends: 1) The mean estimate of Pb in residential soils is three times higher for urbanized areas than non-urbanized areas; 2) The mean estimate of Pb in produce reported in the literature is approximately three orders of magnitude greater than commercially-sourced raw produce monitored in national surveys; 3) The mean estimate of Pb in soils from shooting ranges is two times greater than non-residential Pb contaminated Superfund sites reported in the literature; 4) Research reporting environmental Pb concentrations for school and daycare sites is very limited; 5) Inconsistent sample collection and reporting of results limited synthesis efforts; and 6) The U.S. EPA's Air Quality System was the most robust, publicly available national survey resource. Results from these analyses will inform future multimedia Pb exposure assessments and be useful in prioritizing future research and program development.

Use of chlorine dioxide to sterilize medium for tissue culture of potato.

In vitro cultured seedlings or microtubers are the major starting materials for the production of potato. Currently, seedlings are cultured in media sterilized by autoclaving, which, however, consumes more electricity and takes longer for sterilization, and also requires high temperature-tolerant vessel materials. In order to identify alternative methods of sterilizing culture conditions, the disinfection effects of chlorine dioxide (CD) at 88.0, 29.3, 17.6, 12.6 and 8.8 µM were evaluated in potato medium and vessels. The ≥12.6 µM gaseous CD effectively disinfected vessel through a 30-min fumigation process, and its aqueous solution disinfected potato medium efficiently as well. In presence of 12.6 µM CD in the medium, the potato seedlings had similar morphological features as those grown on autoclaved medium, with some exceptions. The use of 12.6-29.3 µM aqueous CD to sterilize the medium increased antioxidant enzyme activities in potato seedlings, while the use of higher concentration decreased antioxidant enzyme activity levels. SSR analysis did not reveal significant molecular differences in potato seedlings cultured between autoclaved and CD-sterilized medium. In addition to this, CD-sterilized medium induced potato microtuber formation at a similar rate as autoclaved medium. In summary, using CD to sterilize potato medium and vessels did not compromise the growth of seedlings and microtuber induction. This study provides an economical and simplified sterilization method for media used to culture potato plantlets, and this can improve energy use of the large-scale tissue culture industry.

[Study of heterologous efficient synthesis of ß-amyrin and high-density fermentation].

In this study, the synthetic pathway of ß-amyrin was constructed in the pre-constructed Saccharomyces cerevisiae chassis strain Y0 by introducing ß-amyrin synthase from Glycyrrhiza uralensis, resulting strain Y1-C20-6, which successfully produced ß-amyrin up to 5.97 mg·L~(-1). Then, the mevalonate pyrophosphate decarboxylase gene(ERG19), mevalonate kinase gene(ERG12), 3-hydroxy-3-methylglutaryl-CoA synthase gene(ERG13), phosphomevalonate kinase gene(ERG8) and IPP isomerase gene(IDI1)were overexpressed to promoted the metabolic fluxto the direction of ß-amyrin synthesis for further improving ß-amyrin production, resulting the strain Y2-C2-4 which produced ß-amyrin of 10.3 mg·L~(-1)under the shake flask fermentation condition. This is 100% higher than that of strain Y1-C20-6, illustrating the positive effect of the metabolic engineering strategy applied in this study. The titer of ß-amyrin was further improved up to 157.4 mg·L~(-1) in the fed-batch fermentation, which was almost 26 fold of that produced by strain Y1-C20-6. This study not only laid the foundation for the biosynthesis of ß-amyrin but also provided a favorable chassis strain for elucidation of cytochrome oxidases and glycosyltransferases of ß-amyrin-based triterpenoids.

The light-induced transcription factor FtMYB116 promotes accumulation of rutin in Fagopyrum tataricum.

Tartary buckwheat (Fagopyrum tataricum) not only provides a supplement to primary grain crops in China but also has high medicinal value, by virtue of its rich content of flavonoids possessing antioxidant, anti-inflammatory, and anticancer properties. Light is an important environmental factor that can regulate the synthesis of plant secondary metabolites. In this study, we treated tartary buckwheat seedlings with different wavelengths of light and found that red and blue light could increase the content of flavonoids and the expression of genes involved in flavonoid synthesis pathways. Through coexpression analysis, we identified a new MYB transcription factor (FtMYB116) that can be induced by red and blue light. Yeast one-hybrid assays and an electrophoretic mobility shift assay showed that FtMYB116 binds directly to the promoter region of flavonoid-3'-hydroxylase (F3'H), and a transient luciferase activity assay indicated that FtMYB116 can induce F3'H expression. After transforming FtMYB116 into the hairy roots of tartary buckwheat, we observed significant increases in the content of rutin and quercetin. Collectively, our results indicate that red and blue light promote an increase in flavonoid content in tartary buckwheat seedlings; we also identified a new MYB transcription factor, FtMYB116, that promotes the accumulation of rutin via direct activation of F3'H expression.

Comparative transcriptome analysis of roots, stems and leaves of Isodon amethystoides reveals candidate genes involved in Wangzaozins biosynthesis.

BACKGROUND: Isodon amethystoides (Ben-th) Cy Wu et Hsuan is an important traditional medicinal plant endowed with pharmacological properties effective in the treatment of various diseases, including pulmonary tuberculosis. The tetracyclic diterpenoids, Wangzaozins (Wangzaozin A, glaucocalyxin A, glaucocalyxin B), are the major bioactive compounds of I. amethystoides. However, the molecular information about the biosynthesis of these compounds still remains unclear. RESULTS: An examination of the accumulated levels of Wangzaozins in I. amethystoides revealed considerable variations in the root, stem, and leaf tissues of this plant, indicating possible differences in metabolite biosynthesis and accumulation among various tissues. To better elucidate the tetracyclic diterpenoid biosynthesis pathway, we generated transcriptome sequences from the root, stem, and leaf tissues, and performed de novo sequence assembly, yielding 230,974 transcripts and 114,488 unigenes, with average N50 lengths of 1914 and 1241 bp, respectively. Putative functions could be assigned to 73,693 transcripts (31.9%) based on BLAST searches against annotation databases, including GO, KEGG, Swiss-Prot, NR, and Pfam. Moreover, the candidate genes involving in the diterpenoid biosynthesis, such as CPS, KSL, were also analyzed. The expression profiles of eight transcripts, involving the tetracyclic diterpenoid biosynthesis, were validated in different I. amethystoides tissues by qRT-PCR, unraveling the gene expression profile of the pathway. The differential expressions of ISPD, ISPF and ISPH (MEP pathway), and IaCPS and IaKSL (diterpenoid pathway) candidate genes in leaves and roots, may contribute to the high accumulation of Wangzaozins in I. amethystoides leaves. CONCLUSION: The genomic dataset and analyses reported here lay the foundations for further research on this important medicinal plant.

[Rapid identification of Gekko gecko by loop-mediated isothermal amplification based on 12S rRNA sequence].

Gekko gecko (Tokay Gecko) is a valuable traditional Chinese medicine. In this study, the loop-mediated isothermal amplification (LAMP) technique was introduced for visual rapid identification of G. gecko from adulterants. A total of sixty-five 12S rRNA sequences of fourteen species of G. gecko and its adulterants were obtained. The results showed that G. gecko could be identified from its adulterants through BLAST analysis based on 12S rRNA regions. The 12S rRNA sequences of ten batches of G. gecko were conserved. There were only two haplotypes and three variation sites in the available regions for primers design. Six specific LAMP primers were successfully designed online based on 12S rRNA sequences. The visual rapid detection of G. gecko could be achieved with the optimized conditions (64 °C for 1 h and 80 °C for 5 min). And the required minimal template concentration was 5 µg·L⁻¹ while conventional PCR with 0.5 mg·L⁻¹. Consequently, the LAMP method established from this study was rapid, specific, highly sensitive, and simple. It could be applied to detect G. gecko from its adulterants efficiently.

Efficient production of succinic acid from herbal extraction residue hydrolysate.

In this study, six different herbal-extraction residues were evaluated for succinic acid production in terms of chemical composition before and after dilute acid pretreatment (DAP) and sugar release performance. Chemical composition showed that pretreated residues of Glycyrrhiza uralensis Fisch (GUR) and Morus alba L. (MAR) had the highest cellulose content, 50% and 52%, respectively. Higher concentrations of free sugars (71.6 g/L total sugar) and higher hydrolysis yield (92%) were both obtained under 40 FPU/g DM at 10% solid loading for GUR. Using scanning electron microscopy (SEM), GUR was found to show a less compact structure due to process of extraction. Specifically, the fibers in pretreated GUR were coarse and disordered compared with that of GUR indicated by SEM. Finally, 65 g/L succinic acid was produced with a higher yield of 0.89 g/g total sugar or 0.49 g/g GUR. Our results illustrate the potential of GUR for succinic acid production.

[Investigation and optimization on ability of enzymatic hydrolysis of Mori Cortex residue].

Residue of Mori Cortex was studied to optimize its enzymatic hydrolysis process, and explore its potential as a carbon source for biochemistry and biofuel production. The cellulose content of diluted acid pretreated (DAP) and non-pretreated from Mori Cortex were measured in this study, and the results showed that the cellulose content of DAP and non-pretreated from Mori Cortex were 52.5% and 47%, respectively. This higher cellulose content indicated that residue of Mori Cortex had the potential to act as a carbon source for biochemistry and biofuel production. Enzymatic hydrolysis of pretreated and non-pretreated from Mori Cortex was conducted under different enzyme loading amount. 40 FPU·(g DW）⁻¹ enzyme loading was determined as the optimal amount by comparing the yield of sugar and the rate of enzymolysis. Under this condition, the concentrations of glucose, xylose, arabinose sugar were 23.82, 4.84, 3.6 g·L⁻¹, and the corresponding enzymatic hydrolysis rate was 45.33% which was 2.3 times higher than that of non-pretreated from Morus alba residues. Fed-batch enzymatic hydrolysis was conducted finally to get higher sugar yield, and the final glucose concentration reached up to 38 g·L⁻¹ with the enzymatic hydrolysis rate of 36.19%. The results indicated that Mori Cortex residue had higher cellulose and hemicellulose contents, so it had the potential to become a carbon source to produce the bio-chemicals and biofuels. Through enzymatic hydrolysis, it can be converted into microbial available monosaccharides; and through fermentation, it can be converted into high value-added chemicals, biofuels, etc., to solve the problem of residue pollution, and achieve the sustainable development and greening of Chinese pharmaceutical production process.

[Influence of light on gene expression of key synthesis enzyme genes FtANR and FtLAR about proanthocyanidin in seeds of homologous plant of food and medicine Fagopyrum tataricum].

Tartary buckwheat Fagopyrum tataricum is an important medicinal and functional herb due to its rich content of flavonoids in the seeds. F.tataricum exhibited good functions for free radicals scavenging， anti-oxidation， anti-aging activities. Although much genetic knowledge of the synthesis， regulation， accumulation of rutin， the genetic basis of proanthocyanidins(PAs) in tartary buckwheat and their related gene expression changes under different lights(blue， red， far red， ultraviolet light) remain largely unexplored. In this study， we cloned one anthocyanidin reductase gene(ANR) and two leucocyanidin reductase gene(LAR) named FtANR，FtLAR1，FtLAR3 involved in formation of(+)-catechin and(-)-epicatechin precusor proanthocyanidin by digging out F. tataricum seed transcriptome data. The expression data showed that the opposite influence of red light on these gene transcript level compared to others lights. The expression levels of FtANR and FtLAR1 decreased and FtLAR3 appeared increment after exposed in the red light， while the expression levels of those genes appeared opposite result after exposed in the blue and far red light.

[Identification of bear bile powder and its adulterants by using DNA barcoding technology].

To identify the precious bile powder and its adulterants by DNA barcoding, and establish its standard experimental process to ensure the safe and effective utilization. Total twelve sequences from samples of bear bile powder which come from Ursus thibetanus for DNA extraction, PCR(polymerase chain reaction) and sequence, then using CodonCode Aligner V 7.0.1 shear primer region to obtain COI sequence. The COI sequences of U. arctos and their adulterants were obtained from GenBank. MEGA7.0 software was applied for analyzing mutation, calculating intraspecific and interspecific K2P(Kimura 2-Parameter) genetic distance and constructing the Neighbor-joining tree(NJ). The results showed that the maximum K2P genetic distance of bear bile powder of U. thibetanus and U. arctos are far less than minimum K2P genetic distance within its adulterants species, and the results of NJ tree demonstrated that each species could be distinguished from the counterfeits obviously. DNA barcoding is a safe, convenient and reliable technique for species identification, and it is important to establish the standard sequence of COI sequences for animal medicines.