目的 建立了基于核基因内部转录间隔区(ITS2)序列及二级结构鉴别维吾尔药材黑加仑及其混伪品(黑加仑葡萄和异果小檗)的方法。方法 对维吾尔药材黑加仑及其混伪品的ITS2序列进行聚合酶链式反应(PCR)扩增并双向测序,Codon Code Aligner软件对测序峰图进行序列拼接,用MEGA 6.0软件对拼接后的序列进行多重比对。计算种内、种间遗传距离,构建邻接法(Neighbor-Joining,N-J)系统聚类树,预测其ITS2二级结构。结果 经PCR扩增测序后,黑加仑药材ITS2序列长度均为238 bp,GC含量为57.14%~57.98％,A1为主体单倍型;黑加仑葡萄药材序列长度均为226 bp,GC含量为47.79％,仅为一个单倍型;异果小檗药材序列长度均为223 bp,GC含量为53.36％,仅为一个单倍型。并且维吾尔药材黑加仑的种内遗传距离明显小于种间遗传距离。二级结构表明黑加仑及混伪品其螺旋区的茎环数目、大小、位置以及螺旋角度均有明显差异。结论 ITS2序列可以作为鉴定维吾尔药材黑加仑及其混伪品的DNA条形码,为维吾尔药材黑加仑的用药安全提供有效的科学技术手段。
Abstract
OBJECTIVE To identify the Uighur medicine Ribes nigrum and its adulterants(Vitis vinifera and Berberis heteropoda) based on the ITS2 sequence. METHODS The ITS2 sequences of the Uighur medicine Ribes nigrum and its adulterants were subjected to PCR amplification and directional sequencing. CodonCode Aligner software was used to splice sequence of the sequence peak maps, and the MEGA 6.0 software was used to perform multiple alignment of the assembled sequences. In addition, the genetic distances of interspecies and intraspecies were calculated, the NJ system clustering tree was constructed, and ITS2 secondary structure of Uighur medicine Ribes nigrum and its adulterants was predicted. RESULTS The results showed that after PCR amplification and sequencing,the sequence lengths of Ribes nigrum were all 238 bp, and the GC content was 57.14%-57.98%. The sequence lengths of Vitis vinifera were all 226 bp, and the GC content was 47.79%. And the sequence lengths of Berberis heteropoda were all 223 bp, and the GC content was 53.36%. In addition, the intraspecific distance of Uighur medicine Ribes nigrum was more less than the interspecific distance.The secondary structure showed that there were significant differences in the number, size, position and helix angle of stems and rings in the helix region between Ribes nigrum and its adulterants. CONCLUSION The ITS2 sequence could be used as a DNA barcode to identify the Uighur medicine Ribes nigrum and its adulterants, which provides an effective scientific and technological reference for the safety of the Uighur medicine Ribes nigrum.
关键词
维吾尔药材 /
黑加仑 /
核基因内部转录间隔区 /
聚合酶链式反应 /
分子鉴别
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Key words
Uighur medicine /
Ribes nigrum /
ITS2 /
PCR amplification /
molecular identification
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中图分类号：
R282
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脚注
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基金
新疆维吾尔自治区公益性科研院所基本科研业务项目资助(KY2020083);国家中医局青年岐黄学者项目资助
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