Barreto Felipe S, Schoville Sean D, Burton Ronald S
Marine Biology Research Division, Scripps Institution of Oceanography, University of California, San Diego, La Jolla, CA, 92037, USA.
Department of Entomology, University of Wisconsin-Madison, Madison, WI, 53706, USA.
Mol Ecol Resour. 2015 Jul;15(4):868-79. doi: 10.1111/1755-0998.12359. Epub 2014 Dec 26.
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.
反向遗传学工具对于表征新基因的表型以及检验从新一代测序研究中产生的功能假设至关重要。RNA干扰（RNAi）一直是一种广泛使用的技术，通过抑制靶基因的表达来描述或量化其在生理、发育或行为方面的作用。海洋潮间带桡足类动物加州虎斑猛水蚤已成为进化和生理研究的新兴模型，但该物种并不适合大多数基因操作方法。由于甲壳类动物对RNAi介导的基因敲除敏感，我们开发了一种简单的方法来递送基因特异性双链RNA，从而显著抑制靶基因的转录水平。该方案在五个感兴趣的基因上进行了检验，每个基因的表达至少实现了50%的敲除。虽然在任何试验中敲除水平都未达到100%，但对一个热休克基因进行的一项严格对照实验明确表明，这种部分基因抑制可能会导致表型发生显著变化。热休克蛋白β1（hspb1）表达受到抑制的桡足类动物对高温的耐受性显著降低，这证实了先前一项研究所提出的该基因在热应激期间的重要性。这种RNAi方案在加州虎斑猛水蚤中的应用对于研究可能参与生殖隔离、线粒体功能和局部适应的基因的作用将具有重要价值。
