【作者】 查幸福；

【导师】 向仲怀； 夏庆友；

【作者基本信息】 西南大学 ， 特种经济动物饲养， 2006， 博士

【摘要】 家蚕性别决定研究既具有模式研究的科学意义，又具有重要的产业价值，因而一直是蚕业科学的一个重大课题。经典遗传学研究推测家蚕W染色体上Fem基因为家蚕性别决定初级信号，最近家蚕性别决定的下游调控基因Bmdsx也已鉴定。然而，对于从上游的初级信号Fem到下游Bmdsx之间的调控网络，尤其是Bmdsx的上游调控基因，仍然知之甚少。本研究在家蚕全基因组框架图的基础上，应用生物信息学、EST、全基因组芯片分析研究雌雄的差异选择性剪接和雌雄的差异表达，追寻性别决定关键基因，探讨家蚕性别决定分子机制。获得的主要结果如下： 1．家蚕基因组中性别决定同源基因的鉴定 将果蝇所有性别决定基因在家蚕基因组中进行同源性分析，以鉴定家蚕性别决定基因。结果显示，22个性别决定基因在家蚕基因组中具有同源体，并且除Sxl外，其它同源基因都有EST表达证据。此乃首次从家蚕基因组水平大尺度鉴定家蚕性别决定同源基因。 比较分析家蚕与果蝇性别决定调控网络，在性别决定初级信号的基因中，da、emc、gro、sisB、run和dpn在家蚕中具有同源体，而her、sisA和sisC在家蚕中没有同源体，这也许反映了家蚕没有使用X：A作为性别决定初级信号，与经典遗传学研究认为家蚕性别决定初级信号为单一的Fem基因的结果相一致；在剂量补偿途径中，3个基因msl3、mle和mof在家蚕中具有同源体，msl1和msl2基因在家蚕没有同源体，这进一步证明了家蚕缺乏剂量补偿机制；在体细胞性别决定途径中，9个基因Sxl、tra2、dsx、ix、Rbp1、doa、snf、vir和fl(2)d在家蚕中均有同源体，仅tra基因在家蚕中没有同源体，这反映了体细胞性别决定在家蚕中可能较为保守，但个别基因及其调控已发生变化；在求偶行为和种系性别分化途径中，4个基因fru、dsf、ovo和otu在家蚕中都有同源体，表明这两条途径在家蚕中可能很保守。综上述，在性别决定网络中，下游基因大部分在家蚕中存在同源基因，较为保守，而上游基因只有部分基因在家蚕中存在同源基因，变化较大。这从一个方面也证实了Wilkins所提出的性别决定遗传网络由底部向顶部进化的趋势。 2．家蚕性别决定同源基因Bmtar2和Bmix的选择性剪接分析 通过EST与基因组序列比对分析，鉴定了家蚕235个基因的277个选择性剪接体，分析结果表明，选择性剪接在家蚕基因组中广泛地调控生物学功能。在这些基因中，家蚕性别决定重要基因Bmtra2和Bmix也被鉴定具有选择性剪接。 对于Bmtra2基因，鉴定了其的三种mRNA形式，分别命名为Bmtra2-PA、Bmtra2-PB和Bmtra2-PC。Bmtra2-PA编码蛋白与果蝇TRA2有61％的一致性，具有类似的1个RRM结构域和4个RS结构域。基因的外显子与内含子边界位点在物种间也相当保守。Bmtta2-PB与Bmtra2-PA相比，在第2内含子发生了一个可变3’位点类型的选择性剪接，从而Bmtra2-PB编码蛋白比Bmtra2-PA多11个氨基酸残基，这11个氨基酸残基紧邻于第3个RS结构域，这更多还原

【Abstract】 Study on sex determination in Bombyx mori was carried out for understanding the mechanism of sex determination and applying it in sericulture to bring some economic benefit. So study on silkworm sex determination has drawn attention in sericulture all the time. Classical genetics suggested that gene Fem on the w chromosome should be the primary signal in sex determination in Bombyx mori. Recently downstream regulatory gene Bmdsx has been identified. But gene network between genes Fem and Bmdsx is still unknown, especially genes regulating directly Bmdsx. With the finished draft sequence of silkworm genome, mechanism of sex determination in Bombyx mori was explored in our work after we studied different alternative splicing and different expression between male and female silkworm, and found the key genes by analysis of bioinformation, EST and the whole genome microarray. The main results present as follow. 1. Identification of homologs of sex-determining genes in silkworm genomeHomology analysis between the silkworm genome and all the known genes of Drosophila sex determination showed that homologs of 22 genes were found in silkworm. Moreover, their corresponding EST sequences were found except for gene Sxl. Homologs of sex determination were reported firstly after the draft sequence of silkworm genome came out.Analysis on the regulatory network of sex determination between silkworm and Drosophila indicated that, for the primary signal of sex determination, Homologs of genes da, emc, gro, sisB, run and dpn were found in silkworm while no one of genes her, sisA and sisC were found. This suggested that the ratio of X : A was not the primary signal of sex determination in silkworm but the single gene Fem, which was accorded with the point of classical genetics. In the dosage compensation pathway, no homolog of genes msl1 and msl2 were found while the one of the rest three genes msl3, mle and mof were found, which was confirmed that mechanism of dosage compensation is lacking in silkworm. In the pathway of sex determination of somatic cell, homologs of genes Sxl、 tra2、 dsx、 ix、 Rbp1、 doa、 snf、 vir and fl(2)d were found except for tra, which demonstrated that sex determination in somatic cell should be conserved in silkworm though individual gene and its regulation may has changed. In the pathways of sexual behavior and sex determination in the germline, homologs of all the genes fru、 dsf、ovo and otu found. This showed that both of pathways should be conserved in silkworm. In a word, in the regulatory network of silkworm sex determination homologs of most of downstream genes still work and were conserved. Homologs of only part of upstream genes work and something has changed. All this confirmed the idea that sex determination involved from the bottom of the regulatory network to the top proposed by Wilkins.更多还原