| With the improvement of living standard,medicinal materials with both therapeutic and preventive effects have been widely concerned.The authenticity of Chinese medicinal materials and the content of bioactive ingredients are the basis of the quality and efficacy of Chinese medicinal materials.Recently,the adulteration and falsification problems of raw materials in traditional Chinese medicine(TCM)products are serious,which significantly hinders the development of TCM industry and further undermines the worldwide credit of TCM.DNA barcoding has been widely used in the identification of medicinal plants,TCM and crude medicinal slices.However,it is unfit for TCM-based products that go through a various of deep processing leading to serious DNA degradation.To solve this problem,the present thesis applied "nucleotide signature" method,by which means utilizing a short specific sequence with a length of 20 to 50 bp to identify a targeted species in complex formula of TCM-based products.Based on large data volume of ITS2 sequences,nucleotide signatures of Angelica sinensis,Cynomorium songaricum,Cistanche sinensis,Orobanche coerulescens and Boschniakia rossica were developed for the rapid and accurate authentication of Angelicae Sinensis Radix and Cistanches Herba products.(1)Angelicae sinensis radix(Dang Gui)is one commonly applied gynecological drugs.In this section,265 samples of A.sinensis and its adulterants were collected.The ITS2 sequences of these samples were sequenced.Based on one single nucleotide polymorphism(SNP)site unique to A.sinensis,a nucleotide signature consisting of 37-bp(5’-aatccgcgtc atcttagtga gctcaaggac ccttagg-3’)was developed.Then the nucleotide signature was verified by 573 complete ITS2 sequences representing 72 species of Angelica from GenBank,which demonstrated the nucleotide signature is highly conserved species-specific.Primers(DG01F/DG01R)were specially designed to amplify the nucleotide signature region from processed materials.15 product samples purchased online were analyzed.By seeking the nucleotide signature sequence,7 of 15 were identified as counterfeits.28 batches of Chinese patent medicines containing Dang Gui were analyzed.19 of 28 were found to contain the signature,and adulterants such as Ligusticum sinense,Notopterygium incisum,Angelica decursiva and Angelica gigas were detected in other batches.(2)Cistanches Herba is a precious nutritious from Cistanche deserticola and Cistanche tubulosa.In this section,251 ITS2 regions from experiments and 325 ITS2 regions from NCBI database were collected and analyzed.Four nucleotide signatures within 30～37 bp and six species-specific primers were developed based on SNP sites.Then the method was applied to detect 66 Cistanches Herba products on the market including extracts and Chinese patent medicines.36.4%products were detected to be adulterated and Cy.songaricum was the most common adulterant in the market.The results demonstrated the utility of nucleotide signatures in identifying adulterants in mixtures.Beside the adulteration problem,the quality and efficacy of medicinal materials with homology of medicine and food are also concerned in current researches.To improve the content of secondary metabolites in medicinal ingredients has become one of the research directions.Fritillariae Cirrhosae Bulbus,a traditional antitussive and expectorant medicine in China,is in short supply.Villainous weather further increases the demand of this medicines.Their major bioactive compounds,including imperialine and peimisine,both belong to steroidal alkaloids.So far,the steroidal alkaloid synthesis pathway has not been clearly exploited.Data of genome,transcriptomes and genes related to steroidal alkaloid synthesis pathway urgently needed.(1)Transcriptome of Fritillaria taipaiensis was sequenced for the first time.94,396,694(～11.4 Gb)high quality reads obtained using paired-end Illumina sequencing were de novo assembled into 190,350 transcripts.After functional annotation processed with multiple public databases,a series of genes involved in steroid alkaloid biosynthesis,important oxidoreductases and other secondary metabolite pathways including steroid and terpenoid backbone biosynthesis were identified.(2)qRT-PCR was used to verify enzyme genes related to steroid alkaloid synthesis,and the results suggested that these candidates were involved in tissue specific expression.According to the expression analysis,bulbs are more likely to be the main site of the upstream biosynthesis pathway of steroidal alkaloids compared with leaves.And it was speculated that the downstream reactions,including the oxidation-reduction reaction catalyzed by CYPs,may be also mainly reacted in bulbs.(3)In this section,expression vectors of five key enzymes(HMGS,HMGR,DXS,ispH,CAS)related to the steroid alkaloid synthesis pathway,candidate CYP450 superfamily enzymes(CYP90B1,CYP90D2),and three oxidoreductases(DWF5,DWF1 and FK)were successfully constructed using Saccharomyces cerevisiae or E.coli.Meanwhile,the protein expression of expression vectors was induced and the effects of different induction conditions on protein expression were discussed.In conclusion,the medicinal materials with both therapeutic and preventive effects were studied from two perspectives in this study.We established nucleotide signatures for Angelica sinensis and four adulterants of Cistanches Herba.Results demonstrated this method can realize the rapid,accurate,efficient and low-cost identification for TCM products and extend the application range of DNA barcoding technique.Comprehensive transcriptome dataset will serve as a resource for identification of potential candidates,genetic manipulation of targeted bioactive metabolites and development of functionally relevant molecular marker to expedite molecular breeding and conservation efforts in F.taipaiensis.Expression vectors successfully constructed in the study will promote the exploitation of steroid alkaloids biosynthesis. |
PDF Full Text Request