1.College of Life Science and Technology,Jinan University;2.Zhuhai KINGZEST Food Co,LtD
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 wasabia)、辣根(Armoracia rusticana)、芥末(mustard)作原料加工而成。本研究利用核糖体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,为该技术用于芥辣类产品质量检测提供参考。本研究以3种植物材料(山葵、辣根、白芥末)为实验材料,提取基因组DNA,用通用引物ITS2_S2进行PCR扩增得到ITS2片段,测序后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0.88～0.171之间,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、芥末和辣根三物种区分开来。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)建树,山葵与近亲西北山嵛菜聚为一支,棕芥末与白芥末、黄芥末聚为一大支,而辣根单独为一支。山葵与近亲西北山嵛菜、辣根、芥末的ITS2序列分析种间K2P遗传距离在0.030~0.105之间,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。
The pungent condiments in the Japanese dishes are mainly made of Eutrema wasabia, Armorica Rusticana and Mustard, which belong to Brassicaceae plants. This study used the internal transcribed spacer II (ITS2) technology to quickly and accurately identify wasabi, mustard and Armorica Rusticana, providing a reference for the technology used in the quality testing of Pungent products. In this study, three plant materials (Eutrema wasabia, Armorica Rusticana and white mustard) were used as 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 wasabi, white mustard and horseradish is between 0.88 and 0.171, which are all more than 0.01, and there are 36 interspecies variation sites, which preliminarily concluded that the we can distinguish the three species of wasabi, mustard and horseradish with ITS2 sequence. In addition, ITS2 sequences of eutrema wasabi and its close relatives, such as eutrema edwardsii, Armorica Rusticana and mustard, were obtained from GenBank. MEGA7.0 was used to analyze interspecies sequence, calculate K2P and build evolutionary tree by Neighbor-joining (NJ). Eutrema wasabi and eutrema edwardsii are gathered into a large branch, in the meantime, brown mustard, white mustard, and yellow mustard are gathered into a large branch and Armorica Rusticana alone. ITS2 sequence analysis of eutrema wasabi and its close relatives, such as eutrema edwardsii, Armorica Rusticana, and mustard showed that the genetic distance of K2P between species was between 0.030 and 0.105, which are all more than 0.01. This study shows that ITS2 technology can be used to identify relative species such as wasabi, Armorica 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.