Data from: Reverse genetics in the tidepool: knockdown of target gene expression via RNA interference in the copepod Tigriopus californicus

Reverse genetic tools are essential for characterizing phenotypes of novel genes and testing functional hypotheses generated from next-generation sequencing studies. RNA interference (RNAi) has been a widely used technique for describing or quantifying physiological, developmental or behavioural roles of target genes by suppressing their expression. The marine intertidal copepod Tigriopus californicus has become an emerging model for evolutionary and physiological studies, but this species is not amenable to most genetic manipulation approaches. As crustaceans are susceptible to RNAi-mediated gene knock-down, we developed a simple method for delivery of gene-specific double-stranded RNA that results in significant suppression of target gene transcription levels. The protocol was examined on five genes of interest, and for each, at least 50% knock-down in expression was achieved. While knock-down levels did not reach 100% in any trial, a well-controlled experiment with one heat-shock gene showed unambiguously that such partial gene suppression may cause dramatic changes in phenotype. Copepods with suppressed expression of heat-shock protein beta 1 (hspb1) exhibited dramatically decreased tolerance to high temperatures, validating the importance of this gene during thermal stress, as proposed by a previous study. The application of this RNAi protocol in T. californicus will be invaluable for examining the role of genes putatively involved in reproductive isolation, mitochondrial function and local adaptation.

反向遗传学工具（Reverse genetic tools）是表征新基因表型、验证下一代测序研究所得功能假说的核心手段。RNA干扰（RNA interference, RNAi）是一类通过抑制靶基因表达，用以描述或量化靶基因生理、发育及行为功能的常用技术。海洋潮间带桡足类加州虎斑猛水蚤（Tigriopus californicus）现已成为进化与生理学研究中的新兴模式生物，但该物种难以适配绝大多数遗传操作手段。由于甲壳类动物对RNAi介导的基因敲低（RNAi-mediated gene knock-down）具有易感性，我们开发了一种简便的靶基因特异性双链RNA（double-stranded RNA, dsRNA）递送方法，可显著抑制靶基因的转录水平。我们针对5个靶基因验证了该实验方案，所有靶基因的表达量均实现了至少50%的敲低。尽管所有实验中均未实现100%的基因敲低，但针对一个热休克基因开展的严格对照实验清晰表明，此类部分基因抑制可引发表型的显著改变。热休克蛋白β1（heat-shock protein beta 1, hspb1）表达受抑制的桡足类，其高温耐受能力大幅下降，验证了该基因在热应激过程中的重要性，这与此前研究的假说一致。将该RNAi实验方案应用于加州虎斑猛水蚤（T. californicus），将为探究推定参与生殖隔离、线粒体功能及局部适应的基因功能提供极为宝贵的研究手段。