喙尾琵甲DNA提取方法比较与AFLP反应体系的建立  

Genomic DNA Extraction Method and AFLP Amplification Reaction System of Blaps rhynchopetera(Coleoptera:Tenebrionidae)

作　　者：赵敏[1] 冯颖[1] 和锐[1] 陈晓鸣[1] 冀焕红[1] 

机构地区：[1]中国林业科学研究院资源昆虫研究所,国家林业局资源昆虫培育与利用重点实验室,云南昆明650224

出　　处：《林业科学研究》2010年第2期234-240,共7页Forest Research

基　　金：国家林业局林业科学技术重点研究项目(2006-65);国家科技支撑计划课题(2006BAD06B07)

摘　　要：采用CTAB法、改进SDS-蛋白酶K法、SDS-饱和氯化钠法和提取试剂盒对喙尾琵甲的肌肉、虫卵和幼虫进行DNA提取,比较了DNA的提取和保存质量,并对AFLP试验的酶切时间、预扩增产物稀释倍数和选择性扩增引物量等关键因子进行试验和优化。结果表明:4种方法对肌肉组织提取的DNA质量都较好;3种传统方法提取的DNA的保存时间长于试剂盒提取的DNA;使用EcoRⅠ/M seⅠ内切酶组合,10 UEcoRⅠ和2 UM seⅠ分两步各酶切2 h,T4连接酶3 h连接后,以20倍稀释的预扩增产物和5 ng/35 ng的E+3/M+3引物量进行选择性扩增,建立了喙尾琵甲AFLP分子标记体系。The study compared the quality of DNA extracting from muscles, eggs and larvae of Blaps rhynchopetera with CTAB method, improved SDS-protease-K method, SDS-NaCl method and DNA extraction Kit. Some key factors affecting the time of digestion, dilution of pre-amplification product and the amount of selective amplification primer were studied and an optimized AFLP reaction system of B. rhynchopetera was established. High quality genomic DNA could be isolated using four methods from this beetle muscles. DNA extracted with traditional extraction method could keep for longer time than DNA extraction Kit did. The optimal reaction time for enzyme digestion （EcoR I/Mse I ） was 2 hours for each. Products of the pre-amplification diluted 20 fold and 5 ng/35 ng （ E ＋ 3/M ＋ 3 ） was the best amount of the selective amplification primer.

关 键 词：喙尾琵甲 基因组DNA DNA提取 AFLP 

分 类 号：Q963[生物学—昆虫学]