用ITS2鉴定芥辣类调味品中山葵、辣根和芥末组分

Identification of Components of Eutrema wasabi , Armoracia rusticana and Mustard in Pungent Condiment by Internal Transcribed Spacer 2 (ITS2)

摘要:

日式饮食中的芥辣类调味品主要以同属十字花科的山葵（Eutrema wasabi ）、辣根（Armoracia rusticana ）、芥末（mustard）做原料加工而成。利用核糖体DNA内部转录间隔区II（Internal transcribed spacer 2，ITS2）技术快速、准确地鉴定出山葵、芥末和辣根，为该技术应用于芥辣类产品质量检测提供参考。以3种植物材料（山葵、辣根、白芥末）为实验材料，提取基因组DNA，通过引物ITS2_S2进行PCR扩增得到ITS2片段，测序结果通过生物信息学分析进行物种鉴定。MEGA7.0（Molecular evolutionary genetics analysis）分析ITS2序列结果表明，山葵、白芥末和辣根K2P（Kimura 2-parameter）遗传距离在0.088～0.171，均大于0.01，种间变异位点有36个，初步推断利用ITS2序列能将山葵、白芥末和辣根3物种区分开来。另外，GenBank中获得山葵以及近亲西北山嵛菜、辣根、芥末的ITS2序列，MEGA7.0进行种间序列分析，计算K2P，并用邻接法（Neighbor -joining，NJ）构建进化树，山葵与近亲西北山嵛菜聚为一支，棕芥末与白芥末、黑芥末聚为一大支，而辣根单独为一支。通过分析ITS2序列，发现山葵与近亲西北山嵛菜、辣根、芥末的种间K2P遗传距离在0.030~0.105，均大于0.01。研究表明，利用ITS2技术可以鉴别山葵、辣根和芥末等近缘物种，此技术可以用于芥辣类调味品特定原料成分鉴定及定量，为食品质量控制和食品安全提供科学依据。

Abstract:

In Japanese diet, the pungent condiments are mainly made from Eutrema wasabi , Armoracia rusticana and mustard, which are in the Brassicaceae family . Eutrema wasabi , Armoracia rusticana and mustard were quickly and accurately identified in the study by the internal transcribed spacer 2(ITS2) technology, providing a reference for the technology used in the quality control of pungent products. In this study, three plant materials, i.e., Eutrema wasabi, Armoracia rusticana and white mustard, were used as the experimental materials to extract the genomic DNA, and the ITS2 sequence was obtained by PCR amplification using the universal primer ITS2_S2. After sequencing, bioinformatics analysis was performed for species identification. MEGA7.0 (Molecular evolutionary genetics analysis 7.0) analysis of ITS2 sequence showed that the K2P genetic distance (Kimura 2-parameter) of Eutrema wasabi , white mustard and horseradish was between 0.088 and 0.171, which were all higher than 0.01, and there were 36 interspecies variation sites, preliminarily inferring that ITS2 sequence could be used to distinguish the three species of Eutrema wasabi , mustard and horseradish. In addition, ITS2 sequences of Eutrema wasabi and its close relatives, such as Eutrema edwardsii, Armoracia rusticana and mustard, were obtained from GenBank. MEGA7.0 was used to analyze the interspecies sequence, calculate K2P and build evolutionary tree by Neighbor-joining (NJ). Eutrema wasabi and Eutrema edwardsii were gathered into a large branch. In the meantime, brown mustard, white mustard, and black mustard were gathered into a large branch. And Armoracia rusticana was alone as a branch. ITS2 sequence analysis of Eutrema wasabi and its close relatives, such as Eutrema edwardsii, Armoracia rusticana and mustard, showed that the genetic distance of K2P between species was between 0.030 and 0.105, which were all higher than 0.01. This study showed that ITS2 technology could be used to identify the relative species such as Eutrema wasabi, Armoracia rusticana and mustard. This technology can be used to identify and quantify the specific raw material ingredients of pungent condiments, providing a scientific basis for food quality control and food safety.