CRISPR/Cas9-mediated PBP1 and PBP3 mutagenesis induced significant reduction in electrophysiological response to sex pheromones in male Chilo suppressalis.
CRISPR/Cas9-mediated PBP1 and PBP3 mutagenesis induced significant reduction in electrophysiological response to sex pheromones in male Chilo suppressalis.
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DOI：
10.1111/1744-7917.12544
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发表时间：
2019-06
期刊：
影响因子：
4
通讯作者：
Dong SL
中科院分区：
文献类型：
--
作者：
Dong XT;Liao H;Zhu GH;Khuhro SA;Ye ZF;Yan Q;Dong SL
信息素结合蛋白（pbp）被认为与性信息素结合并运输到嗅觉神经元树突膜上的嗅觉受体上，因此在性信息素感知中起着至关重要的作用。然而，PBPs的功能很少在体内得到证实。在本研究中，利用CRISPR/Cas9系统敲除PBP基因的昆虫，对最臭名昭著的pypyal害虫之一Chilo suppressalis的两个PBPs （PBP1和PBP3）进行了体内功能表征。首先，通过将PBP‐单导RNA (sgRNA)/Cas9信使RNA直接注射到新产卵的卵中，在注射后24小时诱导了高的靶基因编辑率（用投票卵进行验证），PBP1‐sgRNA注射的卵诱导率为21.3%，PBP3‐sgRNA注射的卵诱导率为19.5%。其次，通过杂交策略，筛选PBP1或PBP3突变体（均含有过早终止密码子）的昆虫，并在G3代中获得纯合突变体。第三，测量突变昆虫对雌性性信息素的触角电信号（EAG）反应。结果，两种PBP突变雄性小鼠EAG反应均显著降低，且PBP1突变体的降低幅度高于PBP3突变体，表明PBP1的作用更重要。最后，讨论了两种PBPs的相对重要性以及sgRNA‐注射可能引起的脱靶效应。本研究为深入了解不同PBP基因在抑制蛾性信息素感知中的作用及相互作用提供了新的思路，也为其他基因和其他蛾种的基因功能研究提供了有价值的方法论参考。
Pheromone‐binding proteins (PBPs) are thought to bind and transport sex pheromones onto the olfactory receptors on the dendrite membrane of olfactory neurons, and thus play a vital role in sex pheromone perception. However, the function of PBPs has rarely been demonstrated in vivo. In this study, two PBPs (PBP1 and PBP3) of Chilo suppressalis, one of the most notorious pyralid pests, were in vivo functionally characterized using insects with the PBP gene knocked out by the CRISPR/Cas9 system. First, through direct injection of PBP‐single guide RNA (sgRNA)/Cas9 messenger RNA into newly laid eggs, a high rate of target‐gene editing (checked with polled eggs) was induced at 24 h after injection, 21.3% for PBP1‐sgRNA injected eggs and 19.5% for PBP3‐sgRNA injected eggs. Second, by an in‐crossing strategy, insects with mutant PBP1 or PBP3 (both with a premature stop codon) were screened, and homozygous mutants were obtained in the G3 generation. Third, the mutant insects were measured for electroantennogram (EAG) response to female sex pheromones. As a result, both PBP mutant males displayed significant reduction in EAG response, and this reduction in PBP1 mutants was higher than that in PBP3 mutants, indicating a more important role of PBP1. Finally, the relative importance of two PBPs and the possible off target effect induced by sgRNA‐injection are discussed. Taken together, our study provides a deeper insight into the function of and interaction between different PBP genes in sex pheromone perception of C. suppressalis, as well as a valuable reference in methodology for gene functional study in other genes and other moth species.
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