不同花色白芨四种DNA条形码的序列分析.docx

不同花色白芨四种DNA条形码的序列分析 摘要：采用CTAB法提取2种花色白芨基因组DNA，选用trnH-psbA、rbcL、ITS、LSU D1-D3 4种植物DNA条形码通用引物进行PCR扩增，分析黄花、紫花白芨（Bletilla striata）4种DNA条形码的序列，扩增产物纯化后连接T载体并测序，用DNAMAN软件对测序结果进行多序列比对分析。再根据SNP位点设计引物，利用位点特异性PCR扩增进行鉴定。结果表明，4对植物DNA条形码通用引物均能进行扩增，其中trnH-psbA序列没有发现SNP位点、rbcL序列出现2个SNP位点、ITS序列出现62个SNP位点、LSU D1-D3序列出现3个SNP位点。根据LSU D1-D3序列上的2个SNP位点设计的引物DUAN5（5′-TAGTAACGGCGAGCGAAC-3′）和JDHH（5′-TTATCAGCTTCCTTGCGCCCG-3′）能准确鉴定出黄花白芨，成功获得鉴定黄花白芨的引物序列。 关键词：白芨（Bletilla striata）；DNA条形码；PCR；SNP 中图分类号：R282 文献标识码：A 文章编号：0439-8114（2018）17-0106-05 DOI：10.14088/j.cnki.issn0439-8114.2018.17.027 开放科学（资源服务）标识码（OSID）： Abstract： Genomic DNA was extracted from the two kinds of color of Bletilla striata by using the CTAB method. To analysis the sequence of different flower color of Bletilla striata based on the four kinds of DNA barcode for further identification. Four DNA barcoding markers（trnH-psbA，rbcL，ITS and LSU D1-D3） were selected for PCR. PCR product was ligated into T vector for sequencing. The sequencing results were analyzed by using DNAMAN software. According to the SNP loci of different color of Bletilla striata， the primers were designed for identifying different color of Bletilla striata. Result showed that：two kinds of flower color of Bletilla striata genomic DNA can be amplified based on 4 pairs primers of plant DNA barcode markers and SNP loci were found. None SNP locus was found in trnH-psbA sequence. Two SNP loci were found in rbcL sequence. Sixty-two SNP loci were found in ITS sequence. Three SNP loci were found in LSU D1-D3 sequence. Primer DUAN5（5′-TAGTAACGGCGAGCGAAC-3′） and JDHH（5′-TTATCAGCTTCCTTGCGCCCG-3′） can be used for identifying the yellow flower color of Bletilla striata according to the SNP loci in LSU D1-D3 sequence. The primers were found for identifying the yellow flower color of Bletilla striata. Key words： Bletilla striata； DNA barcode； PCR； SNP 白芨（Bletilla striata）伎疲orchidaceae）白芨属（Bletilla）多年生草本植物，是中国重要的中药材。商品白芨为白芨属白芨假鳞茎制成的干燥块茎，性平味苦，具有敛气、渗痰、止血、消肿等功效，主要用于治疗咳血、吐血、外伤出血、溃疡病