采用巢式PCR-直接测序法分析人参达玛烯二醇合成酶基因单核苷酸多态性

Application of nested PCR and direct sequencing to discover single nucleotide polymorphisms in c DNA sequence of dammaranediol synthase gene of Panax ginseng

目的建立人参皂苷生物合成通路中的关键酶——人参达玛烯二醇合成酶(DS)基因c DNA序列的单核苷酸多态性(SNP)检测方法,为人参属植物DS基因的SNP研究以及人参类药材的品种鉴定提供参考。方法采集不同产地、不同品种和生长年限的人参样品,提取RNA,逆转录为c DNA。采用巢式PCR进行扩增,根据人参DS基因c DNA保守序列设计1组引物用于第1轮PCR,2组DS基因上下游分段引物用于第2轮PCR,扩增产物直接测序。测序结果经BLAST同源性比较后,采用DNAMAN软件进行多重比对分析,以发掘不同样品间DS基因差异位点作为候选SNP。结果 57份人参样本共获得111个符合测序要求的DS基因上下游分段PCR产物,其中测序成功103个,序列经BLAST判定为人参DS基因,经多重比对,发现6个样品存在7个SNP位点。结论建立了巢式PCR-直接测序法发掘人参DS基因cDNA序列的SNP,方法具有特异性好、操作简单、结果准确等优点。可用于检测人参样品的DS基因是否含有SNP及其类型,这可为人参及其相关药材和产品的质量评价新方法研究提供有价值的遗传研究工具和分子标记资源。

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 ofDS gene in P. ginseng with the advantages of good specificity, simple operation, and accurate result. This method can be used to detect ifP. 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.