3种DNA分子标记法联合鉴别草珊瑚及其混伪品

目的采用18 S rRNA基因、ITS2序列、SCAR标记等3种DNA分子标记法鉴别草珊瑚,为其分子鉴定提供依据。方法通过PCR扩增、克隆测序后获得草珊瑚18 S rRNA基因序列,并进行Blast比对;通过PCR扩增、测序并注释后获得草珊瑚ITS2序列,从GenBank上收集混伪品和其他植物的ITS2序列,使用MEGA5.5软件,计算种内种间遗传距离,构建系统聚类树;通过RAPD法获得草珊瑚SCAR分子标记,克隆测序后,设计特异性引物扩增草珊瑚及其混伪品。结果获得的草珊瑚18 S rRNA基因长度为1 820 bp,Blast比对显示草珊瑚与金粟兰科同源性最高,同源性为99%,证明其为草珊瑚的18 S rRNA基因。获得的草珊瑚ITS2序列长度为500 bp,草珊瑚与其混伪品之间的遗传距离为0.190～0.219,混伪品之间的遗传距离为0～0.074,聚类分析显示草珊瑚聚为一支,混伪品聚为一支,与其他植物距离较远。获得草珊瑚的SCAR分子标记,用特异性引物扩增出现草珊瑚特异性产物,混伪品未出现特异性产物。结论 3种分子标记法联合可更有效地鉴别草珊瑚及其混伪品,从而建立一套新的鉴别草珊瑚与混伪品的方法,为其鉴别提供新的思路。

Molecular authentication of Sarcandra glabra and its adulterants using three DNA molecular markers

Objective To identify Sarcandra glabra and its adulterants using three DNA molecular markers including 18 S rRNA gene, ITS2 sequence and SCAR marker, and then provide the basis for its molecular authentication. Methods 18 S rRNA gene sequence of S. glabra was obtained by PCR amplification, cloning and sequencing, and then Blast comparison was made in NCBI. The ITS2 sequence of S. glabra was obtained by PCR amplification, sequencing and annotation in ITS2 Database. In the meanwhile, the ITS2 sequences of adulterants and other plants were collected from GenBank. Using MEGA5.5, the genetic distance was calculated between species and then the ITS2 sequences were aligned to construct a phylogenetic clustering tree. SCAR molecular marker of S. glabra was obtained by RAPD. After cloning and sequencing, specific primers were designed to amplify S. glabra and its adulterants. Results The length of 18 S rRNA obtained in our research was 1 820 bp. Blast comparison revealed that there was 99% homology between S. glabra and Chloranthaceae, which proved to be 18 S rRNA gene of S. glabra. The length of the ITS2 sequence in our research was 500 bp. Genetic distance between S.glabra and its adulterants ranged from 0.190 to 0.219, which was far more than genetic distance among adulterants (0.000-0.074). Cluster analysis showed that S. glabra and its adulterants respectively clustered into a different branch, which was far away from other plants. In our research, we obtained SCAR molecular marker of S. glabra and then a pair of specific primers were designed. Using the pair of specific primers, specific products were amplified from genomic DNA of S. glabra, but no specific products were obtained from that of its adulterants. Conclusion We could authenticate S. glabra and its adulterants effectively with the combination of three molecular markers for establishing a novel method to identify S. glabra and its adulterants, which provides a new idea for the authentication of S. glabra.