Circadian rhythms, which are ubiquitous and adaptive, occur across all species, from microbes to humans, in which they organize and modify behavior and physiology. timeless (tim) is a canonical clock gene. The core composition of the Drosophila melanogaster endogenous circadian clock has been extensively investigated; however, in lepidopteran insects, including Bombyx mori, the mechanism is complicated and little is known regarding the participation of tim in the negative feedback loop responsible for behavioral activities. To arrive at a comprehensive understanding of the role of tim in the B. mori endogenous circadian clock, we exploited the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 gene editing system. We attempted to elucidate the functions of tim in the circadian clock of B. mori using Bmtim mutants. The knockouts affected two circadian behavioral activities: adult emergence and embryo hatching rhythms. Quantitative real-time polymerase chain reaction results confirmed that tim-knockouts induced relative reductions in the expression levels, and thereby the oscillation amplitudes, of Bmper and Bmclk messenger RNAs during both the photophase and scotophase. Additionally, the daily rhythmic expression of Bmdbt was upregulated in the photophase and downregulated in the scotophase in a tim-knockout. Our study reveals that tim is integral to the B. mori circadian clock and may be involved in regulating eclosion and hatching rhythms.

中文翻译：

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基于 CRISPR/Cas9 的敲除表明时钟基因永恒对于调节家蚕的昼夜节律行为节律是必不可少的

昼夜节律无处不在且具有适应性，存在于从微生物到人类的所有物种中，它们在其中组织和修改行为和生理机能。timeless ( tim ) 是一个典型的时钟基因。黑腹果蝇内源性生物钟的核心组成已被广泛研究；然而，在包括家蚕在内的鳞翅目昆虫中，其机制很复杂，而且对于tim参与负责行为活动的负反馈回路知之甚少。全面了解tim在B. mori中的作用内源性生物钟，我们利用成簇的规则间隔短回文重复序列 (CRISPR)/CRISPR 相关蛋白 9 基因编辑系统。我们尝试使用Bmtim突变体阐明tim在B. mori生物钟中的功能。基因敲除影响了两种昼夜节律行为活动：成虫出现和胚胎孵化节律。定量实时聚合酶链反应的结果证实，做为计时中的表达水平相对于诱导降低击倒，并且由此振荡幅度，的Bmper和Bmclk的感光期和暗期期间都信使RNA。此外，Bmdbt的日常节奏表达在感光期上调，并在在暗期下调做为计时淘汰赛。我们的研究表明，tim是B. mori生物钟不可或缺的一部分，可能参与调节羽化和孵化节律。