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Molecular Identification Of 14 Species Of Clematis By DNA Bar Code And Study On The Fingerprints Of Clematis Hexapetala Pall

Posted on:2021-01-22Degree:MasterType:Thesis
Country:ChinaCandidate:Q E MuFull Text:PDF
GTID:2404330602495489Subject:Pharmacy
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Objective:The DNA bar code molecular identification method of 14 plants of the Clematis and the HPLC fingerprint of Clematis hexapetala Pall.were established to provide reference for the study of the quality standard of Clematis.Methods:The method of DNADNA bar code molecular identification was used to extract DNA from the samples,and four sequences of ITS2,PsbA-trnH,matk and rbcL were used for PCR amplification and sequencing.The original sequencing peak map was proofread and spliced,arid removed the primers and low quality regions on both sides of the sequence by CodonCodeAligner 17.0(CodonCodeCo;USA)splicing software.The sequences obtained were comprehensively compared and analyzed,and calculated the intraspecific and interspecific genetic distances by MEGA7.0 software.The intraspecific variation relationships of different sequences were analyzed and compared,and the systematic cluster tree was constructed by adjacent(NJ)method.Finally,14 species of Clematis were identified at the molecular level.The HPLC fingerprint of Clematis hexapetala Pall.was studied,by the evaluation software of HPLC method and "similarity Evaluation system of chromatographic fingerprint of traditional Chinese Medicine(2004A)".The chromatographic conditions were Welchrom ColumnC18(4.6mm×300mm,5 ?m).The mobile phase was methanol/0.2%phosphoric acid solution with gradient elution;Velocity of flow:1.0ml/min;Column temperature 30?;The detection wavelength 330nm;Injection volume 10 ?L.Results:the results of DNA extraction,PCR amplification and sequencing showed that DNA could be successfully extracted from the samples.The four sequences of ITS2,PsbA-tmH,matk and rbcL can amplify the target fragments and obtain high quality sequencing bases and peak maps.According to the results of sequence alignment,genetic distance and phylogenetic tree construction,there are 248 permutation sites in ITS2 sequence,including 113 variation sites;440 permutation sites in PsbA-trnH sequence,including 79 variation sites;788 permutation sites in matK sequence,including 767 variation sites;and 583 permutation sites in rbcL sequence,including 408 variation sites,among the 14 species of Clematis.The average of intraspecific genetic distances of ITS2,PsbA-trnH,matk and rbcL sequences were 0.003,0.008,0.000 and 0.513,respectively.The average of interspecific genetic distances of ITS2,PsbA-tmH,matk and rbc sequences of 14 species of Clematis L.were 0.039,0.062,0.099 and 0.735,respectively.The interspecific distance between PsbA-trnH sequence and matk sequence is small.The interspecific variation is significantly larger than the intraspecific variation,indicating that the DNA bar code is suitable for the molecular identification of Clematis L.species.It can be seen from the phylogenetic tree that ITS2 sequences have homology,while rbcL and PsbA-trnH sequences can identify interspecific species.According to the results of HPLC fingerprint,21 common peaks were identified in 14 batches of Clematis hexapetala Pall.,and 3 common peaks were identified as chlorogenic acid,caffeic acid and hyperin.The similarity of 14 batches of Clematis hexapetala Pall was between 0.819 and 0.990.Conclusion:The technololgy of DNA bar code molecular identification can be used for the identification of Mongolian medicine.ITS2 sequence showed that the genetic relationship between Clematis hexapetala and Clematis manshurica Rupr were relatively related to be grouped into one branch;the genetic relationship between Clematis oxtail and Clematis argentilucida(Levi.et Vant.)W.T.Wang were relatively related to be grouped into one branch;the genetic relationship between Clematis macropetala Ledeb.and Clematis ochotensis(Pall.)Poir.were relatively related to be grouped into one branch;while the genetic relationship between Clematis urophylla Franch.and Clematis purpurea were relatively related to be grouped into one branch.PsbA-trnH sequence can identify Clematis hexapetala Pall.,Clematis henryi Oliv.,Clematis purpurea,Clematis urophylla Franch.,Clematis ochotensis(Pall.)Poir.,Clematis macropetala Ledeb.,and Clematis aethusifolia Turcz..RbcL sequence can identify Clematis hexapetala Pall.,Clematis aethusifolia Turcz.,Clematis henryi Oliv.and Clematis argentilucida(Levl.et Vant.)W.T.Wang,while matK sequence can identify Clematis hexapetala Pall.and Clematis brevicaudata.Therefore,ITS2,PsbA-trnH,rbcL and matK sequences can be used for interspecific identification of 14 species of Clematis.The precision,repeatability and stability of HPLC fingerprint are suitable for the identification and quality evaluation of Clematis hexapetala Pall..
Keywords/Search Tags:Clematis, 14 species of medicinal plants, DNA bar code, Clematis hexapetala Pall., HPLC fingerprint
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