瓜实蝇DNA甲基化的MSAP体系建立与优化

摘要

The melon fly [Bactrocera cucurbitae (Coquillett)] is one of the most important vegetables insect pest in China. However, the DNA methylation in melon fly has not been reported yet. Methylation sensitive amplified polymorphism (MSAP) is an important research tecnnique to analysis the DNA methylation. The MSAP can be established through the enzyme digestion reaction, ligation, PCR amplification system, and primer screening, such as 10 U restriction endonuclease is put in the 20 μL enzyme digestion system, and then react with 600 ng of genome DNA at 37℃ overnight; 1U of T4 ligase is put in the 20 μL ligation system, 50 pmol of HpaⅡ-MspⅠ-adapter and 5 pmol of EcoR I-adapter which react at 16℃ for 12 hours. Diluent ligation products were used in PCR to pre-amplification and selected amplification, the result is tested by silver staining and 6% denaturing polyacrylamide gel electrophoresis. Six pairs of primers which suited for DNA methylation sensitive amplified polymorphism were selected out by this system. The MSAP system provides the technical support for the study of the epigenetics research of melon fly.%瓜实蝇[Bactrocera cucurbitae (Coquillett)]是中国重要的蔬菜害虫,但其DNA甲基化研究尚未见报道。甲基化敏感扩增多态性是研究DNA甲基化的重要技术之一。通过对酶切反应、连接、 PCR扩增和引物筛选等条件优化,建立瓜实蝇MSAP反应体系,即：①20μL酶切体系中加入10 U的限制性内切酶与600 ng基因组DNA,于37℃酶切反应过夜；②20μL连接体系中加入T4连接酶1 U, HpaⅡ-MspⅠ-adapter接头50 pmol, EcoR I-adapter接头5 pmol,并于16℃反应12 h；③连接产物稀释后进行PCR预扩增和选择性扩增,再经6%变性聚丙烯酰胺凝胶电泳和银染检测结果。通过该体系筛选出适用于瓜实蝇基因组DNA甲基化多态性研究的6对引物；瓜实蝇MSAP体系为瓜实蝇的表观遗传学研究提供了技术支持。