表皮生长因子EGF信号通路在家蚕腹节雌雄差异发育中的作用

The domesticated silkworm, Bombyx mori, is an important economic insect and a model of lepidopteran insects. The decipherment of the molecular mechanism of its dimorphism is an important topic of developmental biology, but the related report is few. The fact of the development of female eighth abdominal segment into the chitin plate results into the difference of the number of abdominal segments in two sexes, one more in male adults than in female adults. During researching the function of doublesex gene (dsx) of sex determination in B. mori, the proposer found that the ectopic expression of male splice form of dsx in females resulted into the abnormity of chitin plate, and the expression level of the ligand gene spitz (spi) and the phosphorylation level of extracellular-signal-related kinase (ERK) in the posterior abdomen of transgenic females were markedly higher than that in wild-type females, all of which indicated that the cell fate of eighth abdominal segment might be co-regulated by dsx and epidermal growth factor (EGF) signaling. To further clarify the role of EGF signaling in the different development of abdominal segments of two sexes, the following research program has been drafted: First, the differences will be observed about the development and differentiation of cuticle in the posterior abdominal segments of two sexes from propupal to adult stages, using the mitosis marker and intersegmental membrane marked by fluorescence protein; Second, the function of the ligand gene spi of EGF signaling will be detailedly analyzed by transgene and RNAi in the cell proliferation and development of eighth abdominal segment; Third, the female-specific exons of dsx in the genome of B. mori ovary cell line BmN targeted by TALEN, or the sex-specific DSX proteins will be expressed in vitro and in vivo, and then the binding ability of DSX proteins on the upstream regulatory region of the spi gene will be analyzed to confirm the regulatory relationship between dsx and spi. All the studies will present the possibility of some exciting future avenues of research in silkworm and even insect reproductive and developmental biology.

家蚕是重要的经济昆虫和鳞翅目模式昆虫，揭示其两性异形发生的分子机制是发育生物学研究的重要课题，但相关报道很少。雌蚕第8腹节发育为锯齿板致雌蛾较雄蛾少1个腹节。负责人在研究家蚕性别决定双性基因dsx功能时，曾发现dsx雄剪接体异位表达可致雌蛾锯齿板异常，此时EGF信号通路配体基因spi表达水平及ERK蛋白磷酸化水平都显著提高，暗示雌蚕第8腹节发育命运受双性基因dsx及EGF信号通路共同调节。为阐明EGF信号通路在家蚕腹节雌雄差异发育中的作用，本项目拟首先用细胞有丝分裂标记和节间膜荧光蛋白标记观察预蛹期至蛾期雌雄蚕尾部表皮发育及分化的差异；然后用转基因及RNA干涉分析spi在细胞增殖和第8腹节发育中的作用；最后用TALEN靶向敲除家蚕卵巢细胞系BmN中dsx雌特异外显子，以及体内外分别表达DSX蛋白，分析dsx与spi之间的调控关系。研究结果有望为家蚕乃至昆虫生殖及发育生物学研究提供新的参考。

家蚕雌雄差异发育表型明显，但相关分子机制研究较少。雌蚕第8腹节发育为锯齿板致雌蛾较雄蛾少1个腹节，与雌果蝇较雄果蝇多1个腹节刚好相反，是发育生物学研究的理想材料。双性基因dsx的性别特异表达，是调控家蚕雌雄差异发育的关键。本项目通过注射抑制剂影响EGF信号通路重要蛋白磷酸化水平，观察雌雄发育及交配行为变化；通过敲除dsx基因雌特异外显子改变dsx表达形式，经比较转录组寻找发育相关差异表达基因；细胞表达DSX，通过EMSA实验，检测DSX与EGF信号通路配体基因spi启动子区的结合情况；通过同源基因克隆及其启动子鉴定，寻找家蚕体节及成组织细胞发育特异标记，努力构建体节雌雄差异发育过程观测方法；通过转基因异位表达，验证剩余3个DSX蛋白能否参与家蚕性别发育调控，进而获得所有DSX性别调控功能研究相关实验素材。相关研究有望为蚕蛾体节雌雄差异发育分子机制研究提供基础支撑，为家蚕及至昆虫发育生物学研究提供有用参考。