Fukatsu T
National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, Tsukuba, Ibaraki, Japan.
Mol Ecol. 1999 Nov;8(11):1935-45. doi: 10.1046/j.1365-294x.1999.00795.x.
In attempts to establish a convenient and reliable method for field collection and archival preservation of insects and their endosymbiotic microorganisms for molecular analysis, acetone, ethanol, and other organic solvents were tested for DNA preservability of the pea aphid Acyrthosiphon pisum and its intracellular symbiotic bacterium Buchnera sp. After 6 months' storage, not only the band of high-molecular-size DNA but also the bands of rRNA were well preserved in acetone, ethanol, 2-propanol, diethyl ether and ethyl acetate. Polymerase chain reaction (PCR) assays confirmed that the DNA of both the insects and their symbionts was well preserved in these solvents. In contrast, methanol and chloroform showed poor DNA preservability. When water-containing series of acetone and ethanol were examined for DNA preservability, acetone was apparently more robust against water contamination than ethanol. Considering that most biological materials contain high amounts of water, acetone may be a more recommendable preservative for DNA analysis than ethanol which has been widely used for this purpose. The DNA of various insects could be preserved in acetone at room temperature in good condition for several years. In addition to the DNA of the host insects, the DNA of their endosymbionts, including Buchnera and other mycetocyte symbionts, Wolbachia, and gut bacteria, was amplified by PCR after several years of acetone storage. The RNA and protein of the pea aphid and its endosymbiont were also preserved for several years in acetone. After 2 years' storage in acetone, proteins of A. pisum could be analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting, and the endosymbiotic bacteria were successfully detected by immunohistochemistry and in situ hybridization on the tissue sections.
为了建立一种方便可靠的方法，用于野外采集昆虫及其内共生微生物并进行档案保存，以便进行分子分析，对丙酮、乙醇和其他有机溶剂进行了测试，以考察其对豌豆蚜Acyrthosiphon pisum及其细胞内共生细菌布赫纳氏菌Buchnera sp.的DNA保存能力。储存6个月后，不仅高分子量DNA条带，而且rRNA条带在丙酮、乙醇、异丙醇、乙醚和乙酸乙酯中都保存完好。聚合酶链反应（PCR）分析证实，昆虫及其共生体的DNA在这些溶剂中保存良好。相比之下，甲醇和氯仿的DNA保存能力较差。当检测丙酮和乙醇的含水系列对DNA的保存能力时，丙酮显然比乙醇更能抵抗水污染。考虑到大多数生物材料都含有大量水分，与已广泛用于此目的的乙醇相比，丙酮可能是一种更值得推荐的用于DNA分析的防腐剂。各种昆虫的DNA可以在室温下保存在丙酮中数年，状态良好。除了宿主昆虫的DNA外，经过数年丙酮储存后，其包括布赫纳氏菌和其他菌胞共生体、沃尔巴克氏体以及肠道细菌在内的内共生体的DNA也能通过PCR扩增出来。豌豆蚜及其内共生体的RNA和蛋白质在丙酮中也能保存数年。在丙酮中储存2年后，豌豆蚜的蛋白质可以通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和免疫印迹进行分析，并且通过免疫组织化学和组织切片原位杂交成功检测到了内共生细菌。
