Zhu Dan-Tong, Wang Xin-Ru, Ban Fei-Xue, Zou Chi, Liu Shu-Sheng, Wang Xiao-Wei
Ministry of Agriculture Key Laboratory of Molecular Biology of Crop Pathogen and Insects, Institute of Insect Sciences, Zhejiang University.
Ministry of Agriculture Key Laboratory of Molecular Biology of Crop Pathogen and Insects, Institute of Insect Sciences, Zhejiang University;
J Vis Exp. 2017 Jun 19(124):55809. doi: 10.3791/55809.
Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to study the functions and evolution of bacterial symbionts in their host. As most symbionts cannot be cultured in vitro, methods to isolate an adequate quantity of bacteria for genome sequencing are very important. In the whitefly Bemisia tabaci, a number of endosymbionts have been identified and are predicted to be of importance in the development and reproduction of the pests through multiple approaches. However, the mechanism underpinning the associations remains largely unknown. The obstacle partially comes from the fact that the endosymbionts in whitefly, mostly restrained in bacteriocytes, are hard to separate from the host cells. Here we report a step-by-step protocol for the identification, extraction and purification of endosymbionts from the whitefly B. tabaci mainly by dissection and filtration. Endosymbiont samples prepared by this method, although still a mixture of different endosymbiont species, are suitable for subsequent genome sequencing and analysis of the possible roles of endosymbionts in B. tabaci. This method may also be used to isolate endosymbionts from other insects.
细菌共生体与宿主形成紧密的关系，在大多数情况下会给宿主带来益处。基因组信息对于研究细菌共生体在其宿主中的功能和进化至关重要。由于大多数共生体无法在体外培养，因此分离足够数量的细菌用于基因组测序的方法非常重要。在烟粉虱中，已经鉴定出许多内共生体，并通过多种方法预测它们在害虫的发育和繁殖中具有重要作用。然而，这种关联背后的机制在很大程度上仍然未知。这一障碍部分源于这样一个事实，即烟粉虱中的内共生体大多局限于含菌细胞中，很难与宿主细胞分离。在此，我们报告了一种主要通过解剖和过滤从烟粉虱中鉴定、提取和纯化内共生体的分步方案。通过这种方法制备的内共生体样本，尽管仍然是不同内共生体物种的混合物，但适合用于后续的基因组测序以及分析内共生体在烟粉虱中的可能作用。这种方法也可用于从其他昆虫中分离内共生体。
