Application of nested PCR and direct sequencing to discover single nucleotide polymorphisms in cDNA sequence of dammaranediol synthase gene of Panax ginseng / 中草药

ABSTRACT

Objective: To establish a method for discovering single nucleotide polymorphisms (SNPs) in the cDNA sequence of dammaranediol synthase (DS) gene, a key enzyme in the ginsenoside biosynthesis pathway of Panax ginseng, thus to provide reference for the SNP analysis of DS gene and authentication of ginseng medicinal materials. Methods: P. ginseng samples from various cultivars, production regions, and growth years were collected. Total RNA was extracted and reversely transcribed into cDNA. The cDNA was amplified by nested PCR method to prepare the products for directly sequencing. The sequences were analyzed by BLAST for homology alignment and then by DNAMAN for multiple sequence alignment to explore different loci among different samples, which was regarded as candidate SNP. Results: One hundred and eleven amplicons of the upper or lower half part of DS gene were obtained from 57 samples after nested PCR, and 103 amplicons were successfully sequenced. All of these sequences were confirmed to be the P. ginseng DS gene by BLAST analysis. Seven SNPs from six samples were discovered by multiple sequence alignment analysis. Conclusion: We have developed a method to discover the unknown SNPs from the cDNA sequence of DS gene in P. ginseng with the advantages of good specificity, simple operation, and accurate result. This method can be used to detect if P. ginseng samples contain SNP and its type, and it also provides a valuable tool for the genetic research and molecular markers resources for establishing a new quality evaluation method for P. ginseng and related Chinese matereia medica and its products.