Xu Jun, Chen Shuqing, Zeng Baosheng, James Anthony A, Tan Anjiang, Huang Yongping
Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.
University of Chinese Academy of Sciences, Beijing, China.
PLoS Genet. 2017 Jan 19;13(1):e1006576. doi: 10.1371/journal.pgen.1006576. eCollection 2017 Jan.
Manipulation of sex determination pathways in insects provides the basis for a wide spectrum of strategies to benefit agriculture and public health. Furthermore, insects display a remarkable diversity in the genetic pathways that lead to sex differentiation. The silkworm, Bombyx mori, has been cultivated by humans as a beneficial insect for over two millennia, and more recently as a model system for studying lepidopteran genetics and development. Previous studies have identified the B. mori Fem piRNA as the primary female determining factor and BmMasc as its downstream target, while the genetic scenario for male sex determination was still unclear. In the current study, we exploite the transgenic CRISPR/Cas9 system to generate a comprehensive set of knockout mutations in genes BmSxl, Bmtra2, BmImp, BmImpM, BmPSI and BmMasc, to investigate their roles in silkworm sex determination. Absence of Bmtra2 results in the complete depletion of Bmdsx transcripts, which is the conserved downstream factor in the sex determination pathway, and induces embryonic lethality. Loss of BmImp or BmImpM function does not affect the sexual differentiation. Mutations in BmPSI and BmMasc genes affect the splicing of Bmdsx and the female reproductive apparatus appeared in the male external genital. Intriguingly, we identify that BmPSI regulates expression of BmMasc, BmImpM and Bmdsx, supporting the conclusion that it acts as a key auxiliary factor in silkworm male sex determination.
对昆虫性别决定途径的操控为一系列有益于农业和公共卫生的策略提供了基础。此外，昆虫在导致性别分化的遗传途径中表现出显著的多样性。家蚕（Bombyx mori）作为一种益虫，已被人类养殖了两千多年，最近还成为研究鳞翅目遗传学和发育的模式系统。先前的研究已确定家蚕Fem piRNA是主要的雌性决定因子，而BmMasc是其下游靶点，然而雄性性别决定的遗传情况仍不清楚。在本研究中，我们利用转基因CRISPR/Cas9系统在基因BmSxl、Bmtra2、BmImp、BmImpM、BmPSI和BmMasc中产生了一系列全面的敲除突变，以研究它们在家蚕性别决定中的作用。Bmtra2的缺失导致Bmdsx转录本完全缺失，Bmdsx是性别决定途径中保守的下游因子，并诱导胚胎致死。BmImp或BmImpM功能的丧失不影响性别分化。BmPSI和BmMasc基因的突变影响Bmdsx的剪接，并且雄性外生殖器中出现了雌性生殖器官。有趣的是，我们发现BmPSI调节BmMasc、BmImpM和Bmdsx 的表达，支持了它在家蚕雄性性别决定中作为关键辅助因子的结论。
