ITS序列鉴定西红花与其易混中药材

Molecular identity of Crocus sativus and its misused substitutes by ITS sequence

目的:通过研究比较西红花与其易混中药材ITS序列的差异和规律,建立西红花与其易混药材之间的分子鉴别方法。方法:从正品西红花以及番紫花2个园艺品种中提取总DNA,用ITS序列通用引物扩增,扩增产物克隆测序;从GenBank中获得莲须、菊花、玉米、红花的ITS序列。结果:测得西红花以及番紫花2个园艺品种的ITS全长序列,总长度分别为658,634,633 bp,包括ITS1,5.8S和ITS2区,GenBank注册登记号为DQ094185,DQ224363,DQ224364。西红花和番紫花2个园艺品种的ITS序列相似性均高于91%;番紫花2个园艺品种之间ITS序列的相似性高达99.84%;比较ITS1区序列,正品西红花与其易混中药材的差异性均在46%以上;比较ITS2区序列,正品西红花与其易混中药材的差异性均在41%以上。结论:根据ITS序列特征,能有效区分西红花和其他易混中药材。ITS序列是正品西红花鉴定的有效的分子标记。

Objective： To find the patterns of the rDNA ITS sequence variation of Crocus sativus, Chrysanthemum chanetii, Nelumbo nucifera, Zea mays and Garthamus tinctorius and to establish the molecular biological method for the identification of C. sativus and the others. Method：After the total DNA of Crocus sativus, C. vernus-w and C. vernus-p were extracted, the ITS sequence was amplified by PCR with universal primer of ITS and PCR product was sequenced after purification and cloning. The ITS sequences of Chrysanthemum chanetii, Nelumbo nucifera, Zea mays and Garthamus tinctorius were obtained from GenBank. Result：The complete ITS se- quence of Crocus sativus,C, vernus-w and C. vernus-p, including ITS1 rDNA, 5.8S rDNA, ITS2 rDNA were measured. The GenBank accession No. was DQ094185, DQ224363 and DQ224364 respectively. The similarity of ITS sequence between C. sativus and the two garden species of C. vernus was above 91% ; the identity was 99. 84% between C. vernus-w and C. vernus-p. The range of diversity between C. sativus and other herbs was above 46% based on ITS1 and above41% based on ITS2. Conclusion：C. sativus can be distinguished from misused substitutes by the ITS sequence. The ITS sequence is an available molecular marker for identification of the C. sativus.