Abstract：
As a defense mechanism against transposable elements, the PIWI-interacting RNA (piRNA) pathway maintains genomic integrity and ensures proper gametogenesis in gonads. Numerous factors are orchestrated to ensure normal operation of the piRNA pathway. Spindle-E (Spn-E) gene was one of the first genes shown to participate in the piRNA pathway. In this study, we performed functional analysis of Spn-E in the model lepidopteran insect, Bombyx mori. Unlike the germline-specific expression pattern observed in Drosophila and mouse, BmSpn-E was ubiquitously expressed in all tissues tested, and it was highly expressed in gonads. Immunofluorescent staining showed that BmSpn-E was localized in both germ cells and somatic cells in ovary and was expressed in spermatocytes in testis. We used a binary transgenic CRISPR/Cas9 system to construct BmSpn-E mutants. Loss of BmSpn-E expression caused derepression of transposons in gonads. We also found that mutant gonads were much smaller than wild-type gonads and that the number of germ cells was considerably lower in mutant gonads. Quantitative real-time PCR analysis and TUNEL staining revealed that apoptosis was greatly enhanced in mutant gonads. Further, we found that the BmSpn-E mutation impacted gonadal development and gametogenesis at the early larval stage. In summary, our data provided the first evidence that BmSpn-E plays vital roles in gonadal development and gametogenesis in B. mori.
摘要：
作为一种针对转座因子的防御机制，PIWI相互作用RNA(piRNA)途径维持基因组完整性并确保性腺中适当的配子发生。协调了许多因素以确保piRNA途径的正常运行。Spindle-E（Spn-E）基因是最早显示参与piRNA途径的基因之一。在这项研究中,我们对鳞翅目昆虫模型中的Spn-E进行了功能分析，家蚕.与在果蝇和小鼠中观察到的种系特异性表达模式不同，BmSpn-E在所有测试的组织中普遍表达，它在性腺中高度表达。免疫荧光染色显示，BmSpn-E定位于卵巢生殖细胞和体细胞，并在睾丸精母细胞中表达。我们使用二元转基因CRISPR/Cas9系统来构建BmSpn-E突变体。BmSpn-E表达的缺失导致性腺中转座子的抑制。我们还发现突变性腺比野生型性腺小得多，并且突变性腺中生殖细胞的数量大大降低。实时定量PCR分析和TUNEL染色显示，突变性腺细胞凋亡大大增强。Further,我们发现BmSpn-E突变在幼虫早期影响性腺发育和配子发生。总之,我们的数据提供了第一个证据，表明BmSpn-E在B.mori的性腺发育和配子发生中起着至关重要的作用。
