基于ITS2一级序列和二级结构对土茯苓及混伪品的鉴别研究

Identification of Smilacis Glabrae Rhizoma and Its Adulterants Based on ITS2 Sequence and Secondary Structure

目的:使中药材土茯苓与其混伪品得到有效区分,确保土茯苓药材临床用药安全。方法:采用内转录间隔区2 (ITS2)一级序列联合二级结构信息鉴别土茯苓及其混伪品,收集土茯苓正品及混伪品30条ITS2序列,使用基于隐马尔可夫模型(hidden Markov model,HMM)的方法注释,采用MEGA 7.0比对序列,基于Kimura-2-Parameter模型计算种内、种间的遗传距离,并构建邻接法(neighbor-joining,NJ)系统发育树;通过ITS2 Database预测各品种的ITS2二级结构,利用4Sale软件进行二级结构的比对,运用ProfDistS软件基于距离法构建联合一级序列及其二级结构的剖面邻接(profile neighbor joining,PNJ)系统发育树。结果:土茯苓的平均ITS2序列长度为245 bp,平均碱基G+C占比为82.8%,各物种种间遗传距离均显著大于种内遗传距离,NJ系统发育树和PNJ系统发育树的物种分支关系基本一致,土茯苓与其各种混伪品均分别各自聚为一支,可以有效鉴别土茯苓正品与混伪品。结论:ITS2可以作为鉴定土茯苓及其混伪品的DNA条形码,结合二级结构信息可使基于ITS2一级结构结果得到修正和优化,为中药材土茯苓临床安全应用和质量评价提供了参考。

Objective:To effectively distinguish between Smilacis Glabrae Rhizoma and its adulterants to ensure clinical medication safety.Methods:Smilacis Glabrae Rhizoma and its adulterants were identified by ITS2 sequence combined with secondary structure information.Thirty ITS sequences of Smilacis Glabrae Rhizoma and its adulterants were collected and annotated based on the hidden Markov model(HMM).Sequence alignment was carried out by MEGA 7.0.The intraspecific and interspecific genetic distances were calculated based on Kimura-2-Parameter model,and the neighborjoining(NJ) phylogenetic tree was constructed.The ITS2 secondary structures of each species were predicted by ITS2Database.The secondary structures were compared by 4Sale.The profile neighbor joining(PNJ) evolutionary tree was constructed based on the distance method by ProfDistS.Results:The average ITS2 sequence length of Smilacis Glabrae Rhizoma was 245 bp,and the average G+C content was 82.8%.The interspecific genetic distance was significantly greater than the intraspecific one.The species branching of the NJ tree and the PNJ tree was basically the same.Smilacis Glabrae Rhizoma and its adulterants were separately clustered into one branch,respectively,which indicated that Smilacis Glabrae Rhizoma and its adulterants could be effectively identified.Conclusion:ITS2 can be used as DNA barcode for the identification of Smilacis Glabrae Rhizoma and its adulterants.The results obtained only based on ITS2 primary structure can be corrected and optimized by secondary structure information.This study provides a scientific basis for clinical safety application and quality evaluation of Smilacis Glabrae Rhizoma.