线粒体nad1/b-c及nad5/d-e在秦艽组植物中物种鉴定意义的评价
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摘要
龙胆科龙胆属Gentiana秦艽组Sect.Cruciata多种植物作为中药秦艽及藏药解吉(■)的基原,具有重要的药用价值。在课题组前期品种整理及遗传背景分析基础上,本文分别测定秦艽组10种23个居群69份样品以及外类群椭圆叶花锚Halenia elliptica 3份样品的线粒体nad1基因第2内含子区(nad 1/b-c)以及nad5基因第4内含子区(nad5/d-e)部分序列,对其在秦艽组植物的分子系统发育分析及物种鉴定中的意义进行评价。结果显示:nad1/b-c具一粗壮秦艽G.robusta King ex Hook.f.稳定变异位点;同时,对粗茎秦艽G.crassicaulis Duthie ex Burk.及西藏秦艽G.tibetica King ex Hook.f.有一定物种分辨率;nad5/d-e序列相似度高,仅大叶秦艽G.macrophylla Pall.、黄管秦艽G.officinalis H.Smith及管花秦艽G.siphonantha Maxim.ex Kusnez.存在种内不同基因型。进而基于粗壮秦艽G.robusta King ex Hook.f.nad 1/b-c物种分子标记,设计一特异鉴定引物,建立粗壮秦艽特异性引物-PCR鉴定方法。本工作可为协同进化不完全、遗传背景复杂多样的中藏药高山基原植物DNA条形码的构建提供新思路。
Gentiana section Cruciata(Gentianaceae)is a medicinally important section of herbs,including Chinese traditional medicine Gentianae Macrophyllae Radix and Tibetan herb Jieji.Here,we assess the taxonomic significance using mtDNA nad1/b-c and nad5/d-e sequence data.A total of 144 nad1/b-c and nad5/d-e sequences from11 species within Gentianaceae were obtained,including 138 sequences from 10 species within Gentiana section Cruciata and 6 sequences from Halenia elliptica(outgroup).The results showed that mtDNA nad1/b-c has specieslevel resolution within the section of Cruciata,i.e.the variable in the position 45 "C"could be used as a stable marker locus to distinguish G.robusta from other taxa;the variable in the position 352 and 353 "GA"could distinguish G.crassicaulis and G.tibetica from other taxa within the section.Intraspecies genotype variability was detected in nad1/b-c sequences of G.officinalis and G.siphonantha,respectively.These genotypes could be used as potential DNA barcode.In addition,intraspecies genotype variability was detected in nad5/d-e sequences of G.macrophylla G.officinalis and G.siphonantha,respectively.Based on the stable marker locus,a species-specific PCR protocol was developed using the primer PF to identifying G.robusta in the section.This study could expand the understanding of the diversity of mtDNA nad1/b-c and nad5/d-e in the genus Gentiana,and provide the essence for the species identification within Gentiana section Cruciata
Gentiana section Cruciata(Gentianaceae)is a medicinally important section of herbs,including Chinese traditional medicine Gentianae Macrophyllae Radix and Tibetan herb Jieji.Here,we assess the taxonomic significance using mtDNA nad1/b-c and nad5/d-e sequence data.A total of 144 nad1/b-c and nad5/d-e sequences from11 species within Gentianaceae were obtained,including 138 sequences from 10 species within Gentiana section Cruciata and 6 sequences from Halenia elliptica(outgroup).The results showed that mtDNA nad1/b-c has specieslevel resolution within the section of Cruciata,i.e.the variable in the position 45 "C"could be used as a stable marker locus to distinguish G.robusta from other taxa;the variable in the position 352 and 353 "GA"could distinguish G.crassicaulis and G.tibetica from other taxa within the section.Intraspecies genotype variability was detected in nad1/b-c sequences of G.officinalis and G.siphonantha,respectively.These genotypes could be used as potential DNA barcode.In addition,intraspecies genotype variability was detected in nad5/d-e sequences of G.macrophylla G.officinalis and G.siphonantha,respectively.Based on the stable marker locus,a species-specific PCR protocol was developed using the primer PF to identifying G.robusta in the section.This study could expand the understanding of the diversity of mtDNA nad1/b-c and nad5/d-e in the genus Gentiana,and provide the essence for the species identification within Gentiana section Cruciata
引文
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[18]Wei SJ,Zhao ZL,Ni LH,et al.Taxonomic identification of Tibetan herb Bawo Sebo and its chloroplast genome structure[J].Acta Pharm Sin(药学学报),2018,53:1009-1015.
[19]Yao H,Yang P,Zhou H,et al.Identification of medicinal plant Dendrobium based on the chloroplast psbK-psbI intergenic spacer[J].Acta Pharm Sin(药学学报),2015,50:783-787.
[20]Xu J,Chu Y,Liao BS,et al.Panax ginseng genome examination for ginsenoside biosynthesis[J].GigaScience,2017,6:1-15.
[21]Geng LX,Zheng R,Ren J,et al.Application of new type combined fragments:nrDNA ITS+nad 1-intron 2 for identifica-tion of Dendrobium species of Fengdous[J].Acta Pharm Sin(药学学报),2015,50:1060-1067.
[2]Zhao ZL,Gaawe D,Wang ZT.Identification of medicinal plants used as Tibetan Traditional Medicine Jie-Ji[J].J Ethnopharmacol,2010,132:122-126.
[3]Yang YC.Tibetan Medicine(藏药志)[M].Xining:Qinghai People Press,1991:9-12
[4]Ho TN.Flora Reipublicae Popularis Sinicae vol.62(中国植物志,62卷)[M].Beijing:Science Press,1988:59-75.
[5]Xiong B,Zhao ZL,Ni LH,et al.DNA-based identification of Gentiana robusta and related species[J].China J Chin Mater Med(中国中药杂志),2015,40:4680-4685.
[6]Li XJ,Wang LY,Yang HL,et al.Confirmation of natural hybrids between Gentiana straminea and G.siphonantha(Gentianaceae)based on molecular evidence[J].Acta Bot Yunnan(云南植物研究),2007,29:91-97.
[7]Hu QJ,Peng HC,Bi H,et al.Genetic homogenization of the nuclear ITS loci across two morphologically distinct gentians in their overlapping distributions in the Qinghai-Tibet Plateau[J].Sci Rep,2016,6:34244.
[8]Lu JN,Zhao ZL,Ni LH,et al.Identification of medicinal plants based on mitochondrial DNA sequences[J].Chin Tradit Herb Drugs(中草药),2016,47:1791-1796.
[9]Guo YL,Ge S.The utility of mitochondrial nad 1 intron in phylogenetic study of Oryzeae with reference to the systematic position of Porteresia[J].Acta Phytotax Sin(植物分类学报),2004,42:333-344.
[10]Zhang T,Wang ZT,Xu LS,et al.Application of mitochondrial nad 1 intron 2 sequences to molecular identification of some species of Dendrobium Sw.[J].Chin Tradit Herb Drugs(中草药),2005,36:1059-1062.
[11]Ni LH,Zhao ZL.A morphometric comparison of three closely related species of Gentiana(Gentianaceae),endemic to the region of the Qinghai-Tibet Plateau[J].Botany,2018,96:209-215.
[12]Ni LH,Zhao ZL,Xu HX,et al.Chloroplast genome structures in Gentiana(Gentianaceae),based on three medicinal alpine plants used in Tibetan herbal medicine[J].Curr Genet,2017,63:241-252.
[13]Ni LH,Zhao ZL,Xu HX,et al.The complete chloroplast genome of Gentiana straminea(Gentianaceae),an endemic species to the Sino-Himalayan subregion[J].Gene,2016,577:281-288.
[14]Ni LH,Zhao ZL,Xiong B,et al.A strategy for identifying six species of Sect.Cruciata(Gentiana)in Gansu using DNAbarcode sequences[J].Acta Pharm Sin(药学学报),2016,51:821-827.
[15]Doyle JJ,Doyle JL.A rapid DNA isolation procedure for small quantities of fresh leaf material[J].Phytochem Bull,1987,19:11-15.
[16]Duminil J,Pemonge MH,Petit RJ.A set of 35 consensus primer pairs amplifying genes and introns of plant mitochondrial DNA[J].Mol Ecol Notes,2002,2:428-430.
[17]Kumar S,Stecher G,Tamura K.MEGA7:molecular evolutionary genetics analysis version 7.0 for bigger datasets[J].Mol Biol Evol,2016,33:1870-1874.
[18]Wei SJ,Zhao ZL,Ni LH,et al.Taxonomic identification of Tibetan herb Bawo Sebo and its chloroplast genome structure[J].Acta Pharm Sin(药学学报),2018,53:1009-1015.
[19]Yao H,Yang P,Zhou H,et al.Identification of medicinal plant Dendrobium based on the chloroplast psbK-psbI intergenic spacer[J].Acta Pharm Sin(药学学报),2015,50:783-787.
[20]Xu J,Chu Y,Liao BS,et al.Panax ginseng genome examination for ginsenoside biosynthesis[J].GigaScience,2017,6:1-15.
[21]Geng LX,Zheng R,Ren J,et al.Application of new type combined fragments:nrDNA ITS+nad 1-intron 2 for identifica-tion of Dendrobium species of Fengdous[J].Acta Pharm Sin(药学学报),2015,50:1060-1067.