粉蚧标本保存与DNA提取方法的比较  

Comparison of methods for extracting and preserving the DNA of adult Pseudococcidae specimens

作　　者：魏亦寒 郑斯竹[2] 蔡平[1] 詹国辉[2] 高渊[2] 

机构地区：[1]苏州大学金螳螂建筑与城市环境学院,苏州215123 [2]苏州出入境检验检疫局,苏州215000

出　　处：《应用昆虫学报》2015年第4期925-932,共8页Chinese Journal of Applied Entomology

基　　金：国家"十二五"科技支撑计划课题(2012BAK11B03);国家质检总局科技项目(2014IK023);苏州市地方科技项目(SNG201430)

摘　　要：【目的】探讨适合粉蚧的标本保存及DNA提取方法。【方法】采用改良CTAB法、改良SDS法、Gen Mag Bio动物细胞组织/细胞基因组磁珠法以及Gene JET Genomic DNA纯化试剂盒法4种方法分别对新鲜活体4℃、无水乙醇﹣20℃和无水乙醇4℃3种保存方式且保存一年以上的扶桑绵粉蚧Phenacoccus solenopsis成虫进行DNA提取,并对不同提取方法所获取的DNA纯度与质量浓度进行分析比较验证。【结果】3种保存方式中,新鲜活体效果最好,其次为无水乙醇-20℃。无水乙醇4℃效果和无水乙醇﹣20℃无明显区别,均存在一定程度的降解。对于新鲜活体标本,以CTAB法提取的DNA质量最高,其次为Gen Mag Bio磁珠法和SDS法,Gene JET试剂盒法最差;对于无水乙醇﹣20℃和4℃保存时间较长的标本,磁珠法提取的DNA质量明显优于其余3种方法。【结论】无水乙醇﹣20℃可用于粉蚧长期保存,可满足后续分子研究需要;改良CTAB法对新鲜粉蚧成虫DNA提取效果较好,磁珠法对长时间保存DNA存在一定程度降解的粉蚧成虫效果较好。[Objectives] To find which method of extracting and preserving the DNA of adult Pseudococcidae specimerls produced the best quality genomic DNA. [Methods] Genomic Phenacoacus solenopsis DNA from either fresh living adult specimens, or those that had been preserved for over 1 year in 100% ethanol at - 20℃, or 100% ethanol at 4℃, was extracted using four methods; CTAB, SDS, GenMagBio kit and GeneJET kit, and the quality and purity of the extracted genomic DNA compared. [Results] The best quality genomic DNA was obtained from fresh living specimens. Samples that had been stored in 100% ethanol at - 20℃ or 100% ethanol at 4℃ showed evidence of DNA degradation, there was no significant difference in the quality of the DNA obtained from these two storage methods. For fresh samples, the CTAB method produced the best quality DNA, the GenMagBio kit and SDS were produced the next best, and the GeneJET kit produced the worst. However, for samples that had been preserved in 100% ethanol, the GenMagBio kit was the best extraction method. [Conclusion] Storage in 100% ethanol at - 20℃ is suitable for the long term preservation of adult Pseudococcidae specimens and DNA samples obtained from such specimens are suitable for PCR amplification and sequencing. The CTAB method is best for fresh adult specimens but the GenMagBio kit is the best extraction method for specimens that have been stored in ethanol for a long period, especially if there has been serious DNA degradation.

关 键 词：扶桑绵粉蚧 保存方式 DNA提取 CTAB法 磁珠法 

分 类 号：S433[农业科学—农业昆虫与害虫防治]