<br>等位基因特异性敲除揭示了无组织样蛋白在家蚕幼虫黑色素沉着进化丧失中的作用,Insect Molecular Biology

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等位基因特异性敲除揭示了无组织样蛋白在家蚕幼虫黑色素沉着进化丧失中的作用
Insect Molecular Biology ( IF 2.3 ) Pub Date : 2022-06-25 , DOI: 10.1111/imb.12797
Kenta Tomihara ₁ , Peter Andolfatto ₂ , Takashi Kiuchi ₁

Affiliation

家蚕Bombyx mori及其野生祖先B. mandarina作为驯化进化过程的模型得到了广泛研究。这些物种之间的一个显着差异是家蚕幼虫和成虫黑色素沉着的显着减少。在这里，我们评估了 CRISPR/Cas9 靶向敲除色素相关基因的效率，作为了解它们对家蚕驯化相关黑色素沉着损失的潜在贡献的工具。为了证明在B. mandarina中靶向敲除的功效，我们生成了Yellow-y 的纯合 CRISPR/Cas9 靶向敲除。在Yellow-y敲除突变体中，黑色体色在幼虫、蛹和成虫阶段变得更浅，证实了该基因在黑色素形成中的作用。此外，我们对家蚕×柑橘F ₁杂交个体中的色素相关转录因子进行了等位基因特异性CRISPR/Cas9 靶向敲除，该转录因子表现出柑橘样幼虫色素沉着。 F ₁胚胎中apt-like的B. mandarina等位基因的敲除会导致幼虫背皮上出现白色斑块，而相应的B. mori等位基因的敲除则始终表现出正常的 F ₁幼虫色素沉着。这些结果证明了apt-like对家蚕中黑色素沉着减少的进化的贡献。总之，我们的结果证明了 CRISPR/Cas9 靶向敲除作为了解芽孢杆菌相关性状遗传基础的工具的可行性。森驯化.

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Allele-specific knockouts reveal a role for apontic-like in the evolutionary loss of larval melanin pigmentation in the domesticated silkworm, Bombyx mori

The domesticated silkworm, Bombyx mori, and its wild progenitor, B. mandarina, are extensively studied as a model case of the evolutionary process of domestication. A conspicuous difference between these species is the dramatic reduction in melanin pigmentation in both larval and adult B. mori. Here we evaluate the efficiency of CRISPR/Cas9-targeted knockouts of pigment-related genes as a tool to understand their potential contributions to domestication-associated melanin pigmentation loss in B. mori. To demonstrate the efficacy of targeted knockouts in B. mandarina, we generated a homozygous CRISPR/Cas9-targeted knockout of yellow-y. In yellow-y knockout mutants, black body colour became lighter throughout the larval, pupal and adult stages, confirming a role for this gene in melanin pigment formation. Further, we performed allele-specific CRISPR/Cas9-targeted knockouts of the pigment-related transcription factor, apontic-like (apt-like) in B. mori × B. mandarina F₁ hybrid individuals which exhibit B. mandarina-like larval pigmentation. Knockout of the B. mandarina allele of apt-like in F₁ embryos results in white patches on the dorsal integument of larvae, whereas corresponding knockouts of the B. mori allele consistently exhibit normal F₁ larval pigmentation. These results demonstrate a contribution of apt-like to the evolution of reduced melanin pigmentation in B. mori. Together, our results demonstrate the feasibility of CRISPR/Cas9-targeted knockouts as a tool for understanding the genetic basis of traits associated with B. mori domestication.

更新日期：2022-06-25

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