Development of an RNA interference method in the cladoceran crustacean Daphnia magna
Development of an RNA interference method in the cladoceran crustacean Daphnia magna
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DOI：
10.1007/s00427-011-0353-9
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发表时间：
2011-03-01
影响因子：
2.4
通讯作者：
Watanabe, Hajime
中科院分区：
文献类型：
--
作者：
Kato, Yasuhiko;Shiga, Yasuhiro;Watanabe, Hajime
水蚤是淡水中普遍存在的小型甲壳类动物;几十年来，它们一直是生态学，进化和环境科学的研究对象。为了理解在分子水平上水蚤生物学中积累的数据，已经确定了表达的序列标签和基因组序列。然而，这些发现导致了如何理解新发现的基因的功能的问题。双链RNA（dsRNA）介导的RNA干扰（RNAi）是在非转化物种中实现特异性基因沉默的有用工具。因此，我们建立了一种将外源物质注射到排卵卵中的技术，并开发了一种基于dsRNA的大型蚤RNAi方法。在排卵后立即收集卵，并注射对无远端（Dll）基因特异的dsRNA，该基因在无脊椎动物和脊椎动物的附件发育中起作用。我们发现dsRNA成功地触发了Dll mRNA的降解，这以剂量依赖性方式诱导了第二触角的截短。这种效应是序列特异性的，因为：（1）不相关的dsRNA不诱导任何形态学异常，和（2）两个非重叠的Dll dsRNA产生相同的表型。这是国内首次报道利用RNAi技术在D. magna和新兴的基因组序列一起，将有助于推进水蚤的分子生物学知识。
Daphnids are small crustaceans ubiquitous in fresh water; they have been a subject of study in ecology, evolution, and environmental sciences for decades. To understand data accumulated in daphnid biology at the molecular level, expressed sequence tags and a genome sequence have been determined. However, these discoveries lead to the problem of how to understand the functions of newly discovered genes. Double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) is a useful tool to achieve specific gene silencing in nontransformable species. Hence, we established a technique to inject exogenous materials into ovulated eggs and developed a dsRNA-based RNAi method for Daphnia magna. Eggs were collected just after ovulation and injected with dsRNA specific to the Distal-less (Dll) gene, which functions in appendage development in invertebrates and vertebrates. We found that the dsRNA successfully triggered the degradation of Dll mRNAs, which induced the truncation of the second antenna in a dose-dependent manner. This effect was sequence specific in that: (1) an unrelated dsRNA did not induce any morphological abnormalities and (2) two non-overlapping Dll dsRNAs generated the same phenotype. This is the first report of an RNAi technique in D. magna and, together with the emerging genome sequences, will be useful for advancing knowledge of the molecular biology of daphnids.