目的 基于ITS序列上变异位点设计特异性引物对霍山石斛及其常见混伪品(铁皮石斛和河南石斛)进行有效鉴别。方法 利用最大简约法(MP)和贝叶斯法(BI)构建霍山石斛及常见混伪品的系统发育树,并基于ITS序列上变异位点以霍山石斛为正品设计特异性PCR引物SH-CP9s/SH-CP9a、SH-CP25s/SH-CP25a、SH-CP29s/SH-CP29a,优选扩增条件,用于霍山石斛及其混伪品鉴别。结果 在系统发育树中霍山石斛聚类为一个单系。通过对PCR反应的退火温度、DNA模板用量进行优化,并对不同型号的PCR仪和Taq酶进行考察,分别获得霍山石斛的特异性PCR反应程序。在PCR产物中,正品出现目的条带,而混淆品不出现条带。结论 ITS序列能够作为霍山石斛的DNA鉴别条形码,且设计的特异性引物能够有效地区别霍山石斛与其混伪品。
Abstract
OBJECTIVE To design specific identification primers based on ribosomal DNA ITS sequences and accurate identification of Dendrobium huoshanensis and its adultments.METHODS Phylogenetic tree of D. huoshanense and D. officinale and D. henanense by maximum parsimony (MP) and Bayesian inference (BI) were constructed. Specific primers SH-CP9s/SH-CP9a, SH-CP25s/SH-CP25a, SH-CP29s/SH-CP29a for PCR identification of D. huoshanensis based on mutation sites on ITS sequences were designed.RESULTS In the phylogenetic tree, D. huoshanense and D. officinale and D. henanense were clustered into monophyletic group. The specific PCR reaction procedure of D. huoshanense was obtained by investigating the annealing temperature and the amount of DNA template, and different types of PCR and Taq enzymes. In the PCR product, D. huoshanense appearance of the band, and adultments with no band.CONCLUSION The ITS sequences can be used as DNA Barcoding identification of D. huoshanensis, and identify D. huoshanense and its adultments by the designed primers.
关键词
霍山石斛 /
ITS序列 /
DNA条形码 /
特异性鉴别
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Key words
Dendrobium huoshanense /
ITS sequences /
DNA barcoding /
specific identification
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中图分类号：
R284
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参考文献
[1] Ch.P(2015)Vol Ⅰ(中国药典2015年版.一部)[S] . 2015:208.
[2] LI X F, DENG Y Y, PAN L H, et al. Antioxidant effect of polysaccharide from Dendrobium huoshanense on lens tissue of diabetic cataract rats[J] . Chin Tradit Pat Med (中成药), 2012, 34(3):418-421.
[3] HUANG J, LI S L, ZHAO H W, et al. Protective effects of polysaccharides from Dendrobium huoshanense on CCl4-induced acute liver injury in mice[J] . China J Chin Mater Med (中国中药杂志), 2013, 38(4):528-532.
[4] ZHANG D D, HUANG L, ZHA X Q, et al. Inhibitory effect of Dendrobium huoshanense polysaccharides on human gastric cancer cell growth[J] .J Food Sci Biotechnol (食品与生物技术学报), 2014, 33(5):542-547.
[5] DING C C. Identification of three kinds of Dendrobium in huoshan[J] . China J Chin Mater Med (中国中药杂志),1988,13(9):9-10.
[6] CHEN C W, DAI J, YAO H J, et al. Advances in research on Dendrobium resources and identification[J] . Chin Med J Res Prac(现代中药研究与实践), 2013,27(3):84-87.
[7] LI D Z, GAO L M, LI H T, et al. Comparative analysis of a large dataset indicates that internal transcribed spacer (ITS) should be incorporated into the core barcode for seed plants[J] . PNAS, 2011, 108(49):19641-19646.
[8] RONQUIST F, HUELSENBECK J P. MrBayes 3: bayesian phylogenetic inference under mixed models [J] . Bioinformatics, 2003, 19(12):1572-1574.
[9] SWOFFORD D L. Phylogenetic Analysis Using Parsimony(*and Other Methods)[M] . Version 4.0b10. Sunderland, MA:Sinauer Associates, 2002.
[10] NYLANDER J A A. MRMODELTEST version 2.1. Computer Program Distributed by the Author[M] . Uppsala:Uppsala University, 2004.
[11] YUAN Y, JIANG C, HUANG L Q. Strategy and practice on scene application of molecular identification in Chinese medicine materials [J] . China J Chin Mater Med (中国中药杂志), 2013, 38(16):2553-2555.
[12] LIU F, ZHAO Q, DAI J, et al. Microsatellite analysis of cpDNA in Dendrobium huoshanense and its molecular identification[J] . J Chin Med Mater (中药材), 2016, 39(12):2722-2726.
[13] YE S, DHILLON S, KE X, et al. An efficient procedure for genotyping single nucleotide polymorphisms[J] . Nucleic Acids Res, 2001, 29(17):e88.
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脚注
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基金
安徽高校自然科学研究项目资助(KJ2016SD61);中央财政林业科技推广示范资金项目资助(2016TG05);国家林业行业标准制修订项目资助(2015-LY-152);现代农业产业技术体系建设专项资金资助(CARS-21);安徽高校省级自然科学研究项目资助(KJ2014A279)
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