市售中药材冬葵子和苘麻子ITS2条形码鉴定

Identification of Malvae Semen and Abutili Semen using ITS2 DNA barcode

目的利用ITS2条形码序列对市售中药材冬葵子、苘麻子进行分子鉴定,以保证药材使用的正确性和临床药效。方法收集冬葵子、苘麻子及其3种混伪品样品共87份。对实验样品进行ITS2基因片段扩增并双向测序,利用HMMer注释方法获得ITS2序列,再经Codon Code Aligner 6.0.2拼接,使用MEGA6.0进行种内、种间变异分析,遗传距离计算并构建NJ系统发育树;将所得序列转换成二维码图片,扫描识读后通过中药材DNA条形码数据库进行验证。结果冬葵子和苘麻子外观形态相似;冬葵子ITS2序列长度232 bp;GC量为67.6%;苘麻子ITS2序列长度231 bp,GC量为54.1%,二者均无种内变异。冬葵子和苘麻子ITS2序列比对后长度为233 bp,共存在50个变异位点。鉴定结果显示,收集的56份冬葵子药材中26份均为苘麻子,剩余30份为正品。冬葵子和苘麻子与其他3个混伪品的K2P遗传距离为0.224~0.868,平均距离为0.630,种间遗传距离远大于种内遗传距离。基于ITS2序列构建系统发育树可见冬葵子、苘麻子及其3个混伪品各自单独聚成一支,ITS2序列能够明显将其区分;此外,使用二维DNA条形码扫描可准确验证5个物种。结论 ITS2序列能够成功鉴定冬葵子、苘麻子及其常见混伪品,为种子类药材鉴别提供了新工具;二维码技术与DNA条形码技术的结合可以为药材的基原物种提供准确唯一的二维DNA条形码信息,可更好实现药材市场的信息化监管。

Objective To discriminate Malvae Semen,Abutili Semen and other common adulterants using ITS2 barcode,then guarantee the quality and clinical effect of these medical materials.Methods A total of 87 samples comprising Malvae Semen,Abutili Semen and three common adulterants（Astragali Complanati Semen,Semiaquilegiae Radix and Daturae Metelis Semen）were collected.Genomic DNA sequences were extracted from the samples.ITS2 sequences were amplified and sequenced bi-directionally.These sequences were assembled using Codon Code Aligner 6.0.2.The variable sites,genetic distance and neighbor-joining（NJ）phylogenetic tree were calculated using MEGA 6.0 in accordance with the kimura 2-parameter（K2P）model.The obtained sequences were transformed into chromatic barcode images and two-dimensional DNA barcode.ITS2 sequences were verified after scaning and reading two-dimensional codes based on DNA barcode system.Results The recognition difficulty between Malvae Semen and Abutili Semen was expanded with a lot of similarities on the morphological properties.The length of ITS2 sequences of Malvae Semen and Abutili Semen were 232 and 231 bp,respectively.The GC content of Malvae Semen and Abutili Semen were 67.6% and 54.1%.The intra-specific K2P distances of these two crude drugs were both zero.The length of aligned sequences was 233 bp,and there were 50 mutation sites between Malvae Semen and Abutili Semen sequences.The identification results showed that 26 samples were Abutili Semen and30 samples were Malvae Semen in 56 samples of Malvae Semen collected.The inter-specific K2P distances were 0.224—0.868.The average inter-specific K2P distance was 0.630.The minimum inter-specific K2P distances were all larger than the minimum intra-specific distances.NJ tree of ITS2 regions could divide those five species obviously.Those five species could also be correctly verified using the ITS2two-dimensional codes.Conclusion ITS2 sequence barcode is able to identify Malvae Semen,Abutili Semen and other three common adulterants.It provides the important molecular evidence for the identification of those seed Chinese medicines.The combination of two-dimensional code technology and DNA barcode provide accurate and unique two-dimensional DNA barcode information for the original species of medicinal herbs,it can better realize the information supervision of Chinese medicinal materials market.