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

1.家蚕性别特异性选择性剪接的分子机制Bmdsx是家蚕25号染色体上的一个性别决定基因<th>。利用piggyBac基因转化系统，将含有外显子1-5和内含子2-4的Bmdsx基因导入家蚕胚系，获得了一个含有5'和3'端序列的Bmdsx基因。在转基因家蚕中成功地转录并拼接成性别依赖的mRNA。我们还发现，5'端序列含有雄性特异性剪接所需的顺式元件。在UV交联实验中，蛋白质p40与顺式元件结合。我们进行了RNAi实验，证实了p40基因是家蚕雄体剪接所必需的。2. W染色体上的雌体决定基因，W染色体可能编码一个有效的雌体决定基因，尽管目前还不清楚。我们利用各种染色体畸变将雌性决定子定位到Rikishi区域。虽然在Rikishi区域发现了一些蛋白质编码基因，但在一个近缘物种的W染色体上没有发现它们。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。4.生殖细胞的性分化，特别是卵母细胞的性分化，利用SAGE技术分析了卵母细胞发育过程中与发育阶段相关的基因表达。我们注意到一种新的非编码RNA（Ovachamp）在卵巢中大量表达。Ovachamp的RNAi显示了一个有趣的表型，即：e.未发育的卵母细胞，表明该基因是卵子发生所必需的。

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.