›› 2009, Vol. 52 ›› Issue (3): 345-352.

• 研究论文 • 上一篇    



  • 出版日期:2009-04-14 发布日期:2009-03-20
  • 通讯作者: 翟保平

Highquality genomic DNA extraction from ethanolpreserved lepidopteran adults and its application in microsatellite research

  • Online:2009-04-14 Published:2009-03-20

摘要: 高质量的基因组DNA样品是分子生态学研究的先决条件。本研究目的在于探索从东方粘虫Pseudaletia separata (Walker)成虫自然种群的乙醇保存标本中分离高质量基因组DNA的有效方案。在2 mL微型离心管中进行4种提取方案的实验比较,结果发现采用传统的苯酚抽提方法的2种方案提取腹部中段组织的基因组DNA,样品合格率只有7.69%~40%。但是,如果在苯酚抽提以前加入高浓度盐和十六烷基三甲基溴化铵(CTAB),就会使DNA样品合格率达到68.42%~95.28%,而且DNA平均产量达到5.59~10.04 mg/g,明显高于前者的2.83~5.78 mg/g (统计检验表明,在不同种群中差异显著或不显著)。研究结果还证明腹部组织比胸部组织更适宜提取DNA。对来自一个自然种群的99头东方粘虫DNA合格样品的统计分析表明,DNA提取总量(μg)与组织样品用量(mg)之间存在弱的正相关关系,平均DNA提取量(mg/g)与组织样品用量(mg)之间存在中度负相关关系。总之,在2 mL微型离心管中,用10~20 mg腹部组织,利用CTAB+苯酚抽提方法可以获得高纯度和高含量的基因组DNA样品。用该方案提取的基因组DNA能够顺利地进行微卫星位点的分离和基因分型。

关键词: 鳞翅目 东方粘虫, DNA提取, 微卫星, 基因分型

Abstract: Since genomic DNA of high quality is a crucial prerequisite for molecular ecology studies, we specially detected efficient protocols to isolate highquality genomic DNA from ethanolpreserved Pseudaletia separata adults of several natural populations trapped by light traps in fields of different areas. The results of comparative experiment of 4 DNA isolation protocols in 2 mL microcentrifuge tubes showed that only 7.69%-40% of DNA products were qualified in those isolated with conventional phenol extraction protocols. But the qualified rate of DNA products extracted with CTAB+phenol extraction protocols increased to 68.42%-95.28%. Moreover, 5.59-10.04 mg/g DNA was recovered with CTAB+phenol method, much more than 2.83-5.78 mg/g with conventional phenol extraction protocol (significantly different or not from statistical tests in different populations). An additional investigation revealed that abdominal issue was more suitable for DNA extraction than thoracic issue. Correlation analysis of the data set including 99 qualified DNA samples from a natural population indicated that there was a weakly positive correlation between tissue amount (mg) and DNA total yield (μg), a moderately negative correlation between tissue dose (mg) and average DNA yield (mg/g). In conclusion, CTAB+phenol extraction protocol is an efficient method for extracting highquality and highquantity genomic DNA products from 10-20 mg abdominal tissue in a 2 mL microcentrifuge tube, which has been proved suitable for microsatellite identification and genotyping.

Key words: Lepidoptera, Pseudaletia separata, DNA extraction, microsatellite, genotgping