Molecular mechanism of sex determination in lepidopteran insects-The sex chromosomes regulate sex-specific splicing of dsx homologs.

1.Bmdsx性别特异性选择性剪接的分子机制。Bmdsx是位于家蚕Bombyx mori第25号染色体上的性别决定基因。构建了包含外显子1-5和微型内含子2-4的微型Bmdsx基因，该基因仅包含5'和3'末端序列，并通过基于piggyBac的转化系统将其导入家蚕种系中。微型 Bmdsx 转基因在转基因蚕中成功转录并剪接成性别依赖性 mRNA。我们还发现5'端序列包含雄性特异性剪接所需的顺式元件。在紫外交联实验中，蛋白质 p40 与顺式元件结合。我们进行了RNAi实验，证明p40基因对于Bmdsx的雄性型剪接至关重要。2.W染色体上的雌性决定基因。W染色体应该编码一个对家蚕有效的雌性决定子，尽管这一点尚不清楚。我们利用各种染色体畸变将女性决定因素映射到 Rikishi 区域。虽然在Rikishi区域发现了一些蛋白质编码基因，但在密切相关的物种Bombyx mandarina的W染色体上却没有发现这些基因。3.Z染色体的结构和功能。基因组信息预测Z染色体上有655个蛋白质编码基因。男性神经和肌肉系统的基因往往位于 Z 染色体上。例如，四个生物钟成分中的三个（per、tim、clk、cyc）被映射到Z染色体上。4.生殖细胞的性别分化，特别是卵母细胞的分化。进行SAGE分析来分析卵母细胞发育中阶段依赖性基因表达。我们注意到一种新型非编码 RNA (Ovachamp) 在卵巢中大量表达。 Ovachamp 的 RNAi 显示出一个有趣的表型，即。 e.未发育的卵母细胞，表明该基因对于卵子发生至关重要。

1.Molecular mechanism of sex-specific alternative splicing in Bmdsx.Bmdsx is a sex determining gene located on the 25^<th> chromosome in the silkworm, Bombyx mori. A mini Bmdsx gene containing exons 1-5 and mini intorons 2-4, which contained only the 5' and 3' ends sequences, was constructed and introduced into the germ line of the silkworm by the piggyBac-based transformation system. The mini Bmdsx transgene was successfully transcribed and spliced into the sex-dependent mRNAs in the transgenic silkworms. We also found that the 5' end sequences contained a cis element required for the male-specific splicing. In the UV-cross link experiment, a protein, p40, was bound to the cis element. We performed an RNAi experiment and proved that the p40 gene was essential for the male-type splicing of Bmdsx.2.Female determining gene on the W chromosome.The W chromosome should encode a potent female determinant for the silkworm though it is unknown. We mapped the female determinant into the Rikishi region using various kind of the chromosome aberrations. Although a few protein-coding genes were found in the Rikishi region, they are not found on the W chromosome of a closely-related species, Bombyx mandarina.3.Structure and function of the Z chromosome.The genome information predicted 655 protein-coding genes on the Z chromosome. The genes for nervous and muscular systems in the males tend to locate on the Z chromosome. For example, three of the four circadian clock components (per, tim, clk, cyc) were mapped onto the Z chromosome.4.Sexual differentiation of germ cells, especially differentiation of the oocyte.A SAGE analysis were performed to analyze stage-dependent gene expression in oocyte development. We noticed that a novel non-coding RNA (Ovachamp) was abundantly expressed in the ovary. The RNAi of Ovachamp showed an interesting phenotype, i. e. undeveloped oocytes, suggesting that this gene is essential for oogenesis.