基于ITS2序列及二级结构的维吾尔族药材孜然及其混伪品鉴别OACSTPCD

Objective:Using DNA barcode technique,a molecular method was established to identify Cuminum cyminum L.and its adulterants,fennel and coriander seeds.Methods:The total genomic DNA of 38 batches of Uygur medicine Cuminum cyminum L.and its adulterants was extracted,and the sequences of internal transcribed spacer region 2(ITS2)were amplified by polymerase chain reaction(PCR)and sequenced in both directions.The sequencing results were assembled using CodonCode Aligner software,and the sequence characteristics were analyzed using MEGA software.Intraspecific and intergeneric genetic distances were calculated,and a neighbor-joining(NJ)phylogenetic tree was constructed to predict the secondary structure of ITS2.Results:In the ITS2 sequence of Cuminum cyminum L.,the main haplotype was A1,the sequence length was 226 bp,and the guanine(G)cytosine(C)content was 47.79%-48.23%.In the ITS2 sequence of Foeniculum vulgare Mill,the main haplotype was B1,the sequence length was 224-227 bp,and the GC content was 53.30%-55.36%.In the ITS2 sequence of Coriandrum sativum L.,the main haplotype was C1,the sequence length was 220-227 bp,and the GC content was 56.82%-56.83%.The intraspecific genetic distance of Cuminum cyminum L.was significantly smaller than the interspecific genetic distance.The secondary structure analysis showed that there were significant differences in the size,number,position and helix angle of stem loops in the helical region between Cuminum cyminum L.and its adulterants.Conclusion:As a DNA barcode,ITS2 sequence can stably and accurately distinguish Cuminum cyminum L.from its adulterants,providing a new and effective technical means for ensuring the clinical safety of cumin.