基于DNA条形码ITS2序列的野菊及其易混品鉴定研究
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摘要
利用DNA条形码技术通过ITS2序列分析,建立一种快速鉴定野菊及其易混品的方法。提取22份样品的基因组DNA,对ITS2片段进行PCR扩增、双向测序,获得其ITS2序列信息;并从Gen Bank上下载同科或同属序列14条;运用MEGA7. 0对所有36条ITS2序列进行比对、计算种内种间遗传距离,构建Neighbor Joining(NJ)系统进化树,并比较样本ITS2序列间的二级结构差异。结果显示,野菊、条叶旋覆花、蒲儿根、千里光的种内最大遗传距离远小于种间最小遗传距离,具有明显的条形码间距(barcoding gap),而野菊与药用菊的遗传距离重合; NJ树显示野菊、条叶旋覆花、蒲儿根、千里光单独聚为一支,且多数药用菊与部分野菊聚为一亚支;二级结构显示野菊、条叶旋覆花、蒲儿根、千里光之间的二级结构差异显著,且野菊与药用菊共用A和B 2个二级结构;同时也发现野菊是药用菊进化来源之一。因此,ITS2序列作为DNA条形码能准确、快速的鉴别野菊及其易混品,为其种质资源鉴定及临床用药安全提供了重要依据。
DNA barcode technology was used to establish a rapid identification method of Chrysanthemum indicum based on ITS2 sequences. The total DNA was extracted from 22 collected samples,and the ITS2 sequence was amplified by PCR and sequenced,and the information of ITS2 sequence was obtained. Another 14 items of the same family or the same genus were downloaded from Gen Bank.We aligned all 36 sequences,calculated the intraspecific and interspecific distances,and constructed Neighbor Joining( NJ) phylogenetic tree,using MEGA 7. 0. The difference of the secondary structure between the ITS2 sequences was compared. The results showed that the genetic distance of Ch. indicum and Ch. morifolium was overlapped,but the maximum intraspecific distance was far less than the minimum interspecific distance between and among Ch. indicum and other species,with an obvious barcoding gap. The NJ tree showed that Ch. indicum and Ch. morifolium shared a clade,and most of Ch. morifolium with some Ch. indicum were shared a subclade,while Inula lineariifolia,Sinosenecio oldhamianus and Senecio scandens belonged to one clade separately. ITS2 secondary structures for I. lineariifolia,S. oldhamianus and S. scandens were significantly different enough to identify completely but Ch. indicum and Ch. morifolium shared two secondary structures of A and B. It was proved that Ch. indicum was one of the evolutionary sources of Ch.morifolium. Therefore ITS2 sequence as DNA barcode can identify Ch. indicum and its adulterants accurately and quickly. The study provides an important basis for Ch. indicum for the identification of germplasm resources and the safety of clinical medication.
DNA barcode technology was used to establish a rapid identification method of Chrysanthemum indicum based on ITS2 sequences. The total DNA was extracted from 22 collected samples,and the ITS2 sequence was amplified by PCR and sequenced,and the information of ITS2 sequence was obtained. Another 14 items of the same family or the same genus were downloaded from Gen Bank.We aligned all 36 sequences,calculated the intraspecific and interspecific distances,and constructed Neighbor Joining( NJ) phylogenetic tree,using MEGA 7. 0. The difference of the secondary structure between the ITS2 sequences was compared. The results showed that the genetic distance of Ch. indicum and Ch. morifolium was overlapped,but the maximum intraspecific distance was far less than the minimum interspecific distance between and among Ch. indicum and other species,with an obvious barcoding gap. The NJ tree showed that Ch. indicum and Ch. morifolium shared a clade,and most of Ch. morifolium with some Ch. indicum were shared a subclade,while Inula lineariifolia,Sinosenecio oldhamianus and Senecio scandens belonged to one clade separately. ITS2 secondary structures for I. lineariifolia,S. oldhamianus and S. scandens were significantly different enough to identify completely but Ch. indicum and Ch. morifolium shared two secondary structures of A and B. It was proved that Ch. indicum was one of the evolutionary sources of Ch.morifolium. Therefore ITS2 sequence as DNA barcode can identify Ch. indicum and its adulterants accurately and quickly. The study provides an important basis for Ch. indicum for the identification of germplasm resources and the safety of clinical medication.
引文
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[24]代宝强,李思迪,盛益华,等.金银花及山银花提取物体外溶血与凝集试验比较研究[J].中国现代中药,2016,18(11):1458.
[25]任瑶瑶,江南屏,刘睿颖,等.藏药臭蒿及其近缘种药材的ITS2 DNA条形码鉴别[J].中国中药杂志,2017,42(7):1395.
[26]罗焜,陈士林,陈科力,等.基于芸香科的植物通用DNA条形码研究[J].中国科学:生命科学,2010,40(4):342.
[27] Gao T,Yao H J,Liu C,et al. Identification of medicinal plantsin the family Fabaceae using a potential DNA barcode ITS2[J].J Ethnopharmacol,2010,130(1):116.
[28]豆荣昆,毕振飞,白瑞雪,等.黄花紫堇、多刺绿绒蒿及其同属近缘物种的ITS2条形码鉴定与分析[J].中国中药杂志,2015,40(1):1453.
[29]戴思兰,李文彬.菊花起源的RAPD分析[J].植物学报,1998,40(11):1053.
[2]中国药典.一部[S].2015.
[3]潘红烨,邓海欣,陈周全,等.999感冒灵醇沉工艺得失均衡研究[J].中国中药杂志,2016,41(8):1376.
[4]夏伯候,皮胜玲,周亚敏,等.HPLC研究夏桑菊颗粒配伍成分变化规律[J].中药材,2016,39(8):1813.
[5]陈士林,姚辉,宋经元,等.基于DNA barcoding(条形码)技术的中药材鉴定[J].世界科学技术———中医药现代化,2007,9(3):7.
[6] Miao M,Warren A,Song W,et al. Analysis of the internal tran-scribed spacer 2(ITS2)region of scuticociliates and related taxa(Ciliophora,Oligohymenophorea)to infer their evolution andphylogeny[J]. Protist,2008,159(4):519.
[7]石达理,王孟虎,陈思有,等.基于ITS2序列多种龙胆属植物及药材的DNA条形码鉴定研究[J].中药材,2018(1):80.
[8]张建海,冯彬彬,徐晓玉.N、P、K配施效应模型及对野菊花产量和质量的影响[J].中草药,2013,44(11):1495.
[9]蒋征奎,李晓,罗彬.野菊花挥发油抗炎镇痛作用[J].中国实验方剂学杂志,2015,21(16):124.
[10]施剑明,殷嫦嫦,殷明,等.野菊花总黄酮联合顺铂对人骨肉瘤MG-63细胞抑制作用[J].中成药,2014,36(10):2013.
[11] Fang H L,Guo Q S,Shen H J,et al. Genetic diversity evalua-tion of Chrysanthemum indicum L. by medicinal compounds andmolecular biology tools[J]. Biochem Syst Ecol,2012,41(1):26.
[12] Keller A,Schleicher T,Schultz J,et al. 5. 8S-28S rRNA inter-action and HMM-based ITS2 annotation[J]. Gene,2009,430(1):50.
[13] Xie L,Wang Y W,Guan S Y,et al. Prospects and problems foridentification of poisonous plants in China using DNA barcodes[J]. Biomed Environ Sci,2014,27(10):794.
[14] Saitou N,Nei M. The neighbor-joining method:a new methodfor reconstructing phylogenetic trees[J]. Mol Biol Evol,1987,4(4):406.
[15] Liao B,Chen X,Han J,et al. Identification of commercial Gan-oderma(Lingzhi)species by ITS2 sequences[J]. Chin Med-UK,2015,10(1):22.
[16] Kimura M. A simple method for estimating evolutionary rates ofbase substitutions through comparative studies of nucleotide se-quences[J]. J Mol Evol,1980,16(2):111.
[17] Saitou N,Nei M. The neighbor-joining method:a new methodfor reconstructing phylogenetic trees[J]. Mol Biol Evol,1987,4(4):406.
[18] Felsenstein J. Confidence limits on phylogenies:an approachusing the bootstrap[J]. Evolution,1985,39(4):783.
[19] Koetschan C,Frster F,Keller A,et al. The ITS2 databaseⅢ-sequences and structures for phylogeny[J]. Nucleic AcidsRes,2010,38(Database issue):275.
[20]任琛.橐吾属-垂头菊属-蟹甲草属复合体(菊科-千里光族)的细胞分类学、分子系统学和生物地理学研究[D].北京:中国科学院大学,2012.
[21]田鑫.金沸草基原植物野生资源调查与基本性状分析[D].南京:南京农业大学,2014.
[22]吴斌,吴立军.千里光属植物的化学成分研究进展[J].中国中药杂志,2003,28(2):97.
[23]汪宁,焦福贵,潘鲁敏,等.线叶旋覆花的显微鉴定研究[J].现代中药研究与实践,1998,12(1):6.
[24]代宝强,李思迪,盛益华,等.金银花及山银花提取物体外溶血与凝集试验比较研究[J].中国现代中药,2016,18(11):1458.
[25]任瑶瑶,江南屏,刘睿颖,等.藏药臭蒿及其近缘种药材的ITS2 DNA条形码鉴别[J].中国中药杂志,2017,42(7):1395.
[26]罗焜,陈士林,陈科力,等.基于芸香科的植物通用DNA条形码研究[J].中国科学:生命科学,2010,40(4):342.
[27] Gao T,Yao H J,Liu C,et al. Identification of medicinal plantsin the family Fabaceae using a potential DNA barcode ITS2[J].J Ethnopharmacol,2010,130(1):116.
[28]豆荣昆,毕振飞,白瑞雪,等.黄花紫堇、多刺绿绒蒿及其同属近缘物种的ITS2条形码鉴定与分析[J].中国中药杂志,2015,40(1):1453.
[29]戴思兰,李文彬.菊花起源的RAPD分析[J].植物学报,1998,40(11):1053.