CRISPR/Cas9-Mediated Vitellogenin Receptor Knockout Leads to Functional Deficiency in the Reproductive Development of Plutella xylostella
CRISPR/Cas9-Mediated Vitellogenin Receptor Knockout Leads to Functional Deficiency in the Reproductive Development of Plutella xylostella
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CRISPR/Cas9介导的卵黄蛋白原受体敲除导致小菜蛾生殖发育功能缺陷
DOI：
10.3389/fphys.2019.01585
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发表时间：
2020-01-23
影响因子：
4
通讯作者：
You, Min-Sheng
中科院分区：
文献类型：
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作者：
Peng, Lu;Wang, Qing;You, Min-Sheng
卵黄蛋白原受体（VgR）属于低密度脂蛋白受体（LDLR）基因超家族，在Vg运输、卵黄沉积和卵母细胞发育中发挥着不可或缺的作用。因此，它已成为害虫防治的一个有前途的目标。在十字花科作物的破坏性害虫小菜蛾 Plutella xylostella (L.) 中，VgR 在 Vg 运输和生殖功能中的参与尚不清楚。在此，我们克隆并鉴定了小菜蛾VgR的完整cDNA序列，其编码1805个氨基酸残基，并包含LDLR超家族的4个保守结构域。 PxVgR 主要在成年女性中表达，更具体地说在卵巢中表达。 PxVgR 蛋白也显示出与 PxVgR 转录物相似的表达谱。 CRISPR/Cas9 介导的 PxVgR 敲除产生了 PxVgR 中 5 bp 核苷酸缺失的小菜蛾纯合突变体。在突变个体的卵巢和卵子中检测到PxVgR蛋白的表达缺陷。在突变个体的卵中仍然检测到Vg蛋白，但表达水平降低。然而，PxVg 转录本并未受到 PxVgR 敲除的显着影响。 PxVgR 的敲除会导致新出现的雌性的卵巢变短。野生个体和突变个体在交配后前 3 天内产卵数量没有显着差异。 PxVgR基因的缺失导致蛋更小、更白，孵化率更低。本研究首次利用 CRISPR/Cas9 技术报道了 VgR 在小菜蛾 Vg 运输、卵巢发育、产卵和胚胎发育中的功能。本研究为了解小菜蛾繁殖的分子机制以及利用VgR作为潜在的基于遗传的分子靶点来更好地控制小菜蛾奠定了基础。
The vitellogenin receptor (VgR) belongs to the low-density lipoprotein receptor (LDLR) gene superfamily and plays an indispensable role in Vg transport, yolk deposition, and oocyte development. For this reason, it has become a promising target for pest control. The involvement of VgR in Vg transport and reproductive functions remains unclear in diamondback moths, Plutella xylostella (L.), a destructive pest of cruciferous crops. Here, we cloned and identified the complete cDNA sequence of P. xylostella VgR, which encoded 1805 amino acid residues and contained four conserved domains of LDLR superfamily. PxVgR was mainly expressed in female adults, more specifically in the ovary. PxVgR protein also showed the similar expression profile with the PxVgR transcript. CRISPR/Cas9-mediated PxVgR knockout created a homozygous mutant of P. xylostella with 5-bp-nucleotide deletion in the PxVgR. The expression deficiency of PxVgR protein was detected in the ovaries and eggs of mutant individuals. Vg protein was still detected in the eggs of the mutant individuals, but with a decreased expression level. However, PxVg transcripts were not significantly affected by the PxVgR knockout. Knockout of PxVgR resulted in shorter ovarioles of newly emerged females. No significant difference was detected between wild and mutant individuals in terms of the number of eggs laid in the first 3 days after mating. The loss of PxVgR gene resulted in smaller and whiter eggs and lower egg hatching rate. This study represents the first report on the functions of VgR in Vg transport, ovary development, oviposition, and embryonic development of P. xylostella using CRISPR/Cas9 technology. This study lays the foundation for understanding molecular mechanisms of P. xylostella reproduction, and for making use of VgR as a potential genetic-based molecular target for better control of the P. xylostella.