| Herbgenomics is a discipline that uncovers the genetic information and regulatory networks of herbs to clarify their molecular mechanism in the prevention and treatment of human diseases.Herbgenomics focuses on the interactions between herbal medicines and humans.The main research content of Herbgenomics involves the theoretic system,the key technologies,and the practical applications.The key technologies,such as DNA barcoding,shotgun metagenomic sequencing,and single molecule,real-time(SMRT)sequencing,are applied to establish the medicinal plant DNA barcoding system of Changbai Mountain,and to develop new methods for the quality control of traditional Chinese patent medicine(TCPM).The DNA barcoding identification of traditional Chinese medicine(TCM)needs powerful database as a potent support.To improve the identification ability,the database needs continuous expansion,update,and maintenance.At present,there may be undeclared species contained in TCPM which lead to drug safety issues.However,the quality control methods recorded in the Chinese Pharmacopoeia are difficult to meet the biomonitoring requirements for the biological ingredient composition.Thus,a new method to handle this problem is an urgent need.In this study,the medicinal plants in Changbai Mountain and TCPM are authenticated with DNA barcoding and different sequencing platforms.The main contents and conclusions are as follows:1.Establishment of medicinal plant DNA barcoding system of Changbai Mountain:Changbai Mountain is a comparatively complete preserved ecosystem with abundant biodiversity at the same latitude of northern hemisphere,and also the largest medicinal plant repositories in Northeast Asia.In this study,a total of 1,877 samples,belonging to 678 species,400 genera,and 107 families were collected in Changbai Mountain.Nineteen genera were collected with five or more species.Eight genera of them,including 158 samples belonging to 55 species,were randomly selected to evaluate the identification efficiency of the four most commonly used DNA barcodes(ITS2,psbA-trnH,matK,and rbcL).Results showed that ITS2 and psbA-trnH regions performed better than the other two loci.Thus,these two loci were used to establish the medicinal plant DNA barcoding system of Changbai Mountain.1,736 and 1,665 sequences belonging to ITS2 and psbA-trnH regions were obtained and included in the database,respectively.It is the first medicinal plant DNA barcode database based on a national reserve.It provides public basic information about the medicinal plants distributed in Changbai Mountain and DNA barcoding identification of unknown samples based on the Basic Local Alignment Search Tool(BLAST)algorithm.It will be a powerful tool for the biodiversity conservation of Changbai Mountain National Reserve.Compared with the"DNA barcoding system for identifying herbal medicine",38.3%(264)of the species collected in this study were included in the database for the first time.In addition,this database can provide effective assistance for rountine inspection of customs and food and drug administrations and ensure the drug safety from the source.2.Precise species detection of traditional Chinese patent medicine by shotgun metagenomic sequencing:All ten herbal materials in Longdan Xiegan Wan(LDXGW)were collected and identified to formulate the lab-made reference samples(RF01-02)according to the Chinese Pharmacopeia.In addition,Panax ginseng was used as the positive control in one of the reference sample(RF02).Moreover,three batches of commercial samples(LDXGW01-03)were also included in this study.Genomic DNA was extracted to build the PCR free DNA library.The shotgun metagenomic sequencing was performed on Illumina Hiseq 2500 platform.The shotgun metagenomic sequencing data was cleaned to remove adapters and low-quality leading and trailing sequences,and then individually mapped to the ITS2,psbA-trnH,and matK databases.The mapped reads were assembled and used to select useful DNA barcode sequences through BLAST in the DNA Barcoding System for Identifying Herbal Medicine.Besides,the chemical tests for the commercial samples,including HPLC and TLC,were conducted according to the Chinese Pharmacopoeia.Over 100 G of raw data were obtained.After assembling and filtering all the reads,a total of 261 contigs were gained,which belonged to the ITS2,psbA-trnH,and matK regions.Bioinformatics analysis indicated that the ITS2 region showed the highest identification efficiency.All prescribed species,including the positive control in RF02,could be successfully detected in the lab-made reference samples.The results suggested that the method had favourable sensitivity,stability and accuracy.The commercial samples all met the requirements of the Chinese Pharmacopoeia according to the TLC and HPLC tests.However,the shotgun metagenomic sequencing detected the substitution of Akebiae Caulis(Mutong)in the commercial samples,while the chemical analyses could not distinguish.The results highlight that shotgun metagenomic sequencing is a complementary method for the precise species detection of TCPMs.It is a breakthrough in the biological monitoring of TCPM with complex formulations.3.Biomonitoring for traditional Chinese patent medicine using DNA metabarcoding and single molecule,real-time(SMTR)sequencing:All nine herbal materials in Jiuwei Qianghuo Wan(JWQHW)were collected and identified to formulate the lab-made reference samples(RF01-02)according to the Chinese Pharmacopeia.Similarly,Panax ginseng was added into RF02 as the positive control.Moreover,three batches of commercial samples(JWQHW01-03)were also included in this study.Genomic DNA was extracted to gain the PCR products of ITS2 and psbA-trnH regions.The PacBio RSII SMRT sequencing platform and circular-consensus sequencing(CCS)strategy were used to sequence the ITS2 and psbA-trnH amplicons.The raw data were analyzed to gain the clustered reads.The species identification was performed in the DNA Barcoding System for Identifying Herbal Medicine.A total of 518.6 MB raw data were obtained using SMRT sequencing.Ultimately,5,416 and 4,342 CCS reads were identified as ITS2 and psbA-trnH regions,respectively.Species identification results showed that seven prescribed herbal materials were detected in both references samples,which were consistent with the Sanger sequencing reustls.Furthermore,the positive control was detected in RF02.The results suggest that it is repeatable,reliable,and sensitive enough to detect species in the TCPMs.Rehmanniae Radix and Angelicae dahuricae Radix were not detected in all three commercial samples.Furthermore,Scutellariae Radix was not detected in JWQHW02.In addition to the prescribed species,several other potential contaminant species were detected in commercial samples.The CCS reads mapping to the ITS2 and psbA-trnH regions were classified into 22 families with 47 genera and 12 families with 18 genera,respectively.For instance,Atractylodes macrocephala Koidzumi was found in JWQHW02 and species belonging to Angelica and Peucedanum were found in all commercial samples.This study shows that SMRT sequencing is applicable to the quality control in the whole process of the production and processing of TCPMs,and is a necessary link in the pharmacovigilance of TCPMs,which provides a new idea for improving the quality control method of TCPMs in Chinese Pharmacopoeia. |
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