Honey bee functional genomics using symbiont-mediated RNAi
Honey bee functional genomics using symbiont-mediated RNAi
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DOI：
10.1038/s41596-022-00778-4
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发表时间：
2022-12-02
期刊：
影响因子：
14.8
通讯作者：
Barrick, Jeffrey E.
中科院分区：
文献类型：
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作者：
Lariviere, Patrick J.;Leonard, Sean P.;Barrick, Jeffrey E.
蜜蜂是研究社会行为、发育和认知不可或缺的传粉者和模式生物。然而，它们的真社会性使得难以使用标准的正向遗传学方法来研究基因功能。目前，蜜蜂中的大多数功能基因组学研究利用双链RNA（dsRNA）注射或喂养来诱导RNAi介导的目的基因敲低。然而，dsRNA注射是费力且有害的，并且dsRNA补料难以廉价地规模化。此外，两种方法都需要重复施用dsRNA以确保持续的RNAi应答。为了填补这一空白，我们设计了蜜蜂肠道细菌Sacrifgrassella alvi，以诱导持续的宿主RNA干扰反应，从而减少靶基因的表达。为了使用工程化共生体（FUGUES）程序来应用该功能基因组学，使用Golden Gate组装在大肠杆菌中克隆dsRNA表达质粒，然后转移到S.阿尔维。成年工蜂然后用工程化的S.阿尔维。最后，通过qRT-PCR验证基因敲除，并且可以进一步评估感兴趣的蜜蜂表型。在定殖后5天，整个蜂体中靶基因的表达减少多达50-75%。该方案可由具有微生物学和分子克隆技术的蜜蜂研究人员在4周内完成。Fugues目前提供了一种简化和可扩展的方法来研究蜜蜂的生物学。工程其他微生物共生体，以影响他们的主机的方式是类似于本协议中所描述的，可能会被证明是有用的，在未来研究其他昆虫和动物物种。
Honey bees are indispensable pollinators and model organisms for studying social behavior, development and cognition. However, their eusociality makes it difficult to use standard forward genetic approaches to study gene function. Most functional genomics studies in bees currently utilize double-stranded RNA (dsRNA) injection or feeding to induce RNAi-mediated knockdown of a gene of interest. However, dsRNA injection is laborious and harmful, and dsRNA feeding is difficult to scale cheaply. Further, both methods require repeated dsRNA administration to ensure a continued RNAi response. To fill this gap, we engineered the bee gut bacterium Snodgrassella alvi to induce a sustained host RNA interference response that reduces expression of a targeted gene. To employ this functional genomics using engineered symbionts (FUGUES) procedure, a dsRNA expression plasmid is cloned in Escherichia coli using Golden Gate assembly and then transferred to S. alvi. Adult worker bees are then colonized with engineered S. alvi. Finally, gene knockdown is verified through qRT-PCR, and bee phenotypes of interest can be further assessed. Expression of targeted genes is reduced by as much as 50-75% throughout the entire bee body by 5 d after colonization. This protocol can be accomplished in 4 weeks by bee researchers with microbiology and molecular cloning skills. FUGUES currently offers a streamlined and scalable approach for studying the biology of honey bees. Engineering other microbial symbionts to influence their hosts in ways that are similar to those described in this protocol may prove useful for studying additional insect and animal species in the future.