- 學位論文
解析東方果實蠅 oskar 基因產物在卵後端之聚集與生殖漿的形成
Posterior localization of oskar mRNA and protein evidences the assembly of germ plasm during oogenesis in the oriental fruit fly Bactrocera dorsalis
摘要
生殖細胞為專司遺傳訊息傳遞至子代之細胞。已知部分動物如黑腹果蠅，生殖細胞特化通常發生於發育的早期，其形成需倚賴聚集在卵室後端的細胞質，又稱生殖漿。母系遺傳的生殖細胞決定因子表現於生殖漿，並於胚胎發育時期促使後端形成生殖細胞 (極細胞)。在眾多生殖細胞決定因子中，oskar 基因扮演招攬並固定其他生殖漿組成物於後端之上游角色。oskar 基因產物表現於卵發育與早期胚胎發育的後端，此現象已被證實保守於部分完全變態昆蟲中，如蚊和蜂，因而推論 oskar 基因產物可標定卵發育時期的生殖漿形成。根據推論，我們選殖和黑腹果蠅同屬雙翅目之東方果實蠅 oskar 同源基因 (Bdosk)，並偵測卵發育時期的基因表現。透過整體原位雜合法我們發現 Bdosk mRNA 的表現模式與黑腹果蠅 Dmosk mRNA 相似，皆表現於卵細胞的後端。而螢光免疫染色結果呈現 BdOsk 蛋白表現模式同樣與 DmOsk 蛋白相似，證實 oskar 基因於雙翅目昆蟲中保守參與生殖漿組裝。為了驗證 oskar 基因作為分子錨的保守性，我們透過 UAS-Bdosk-Dmbcd 3’ UTR 轉基因黑腹果蠅，異位表現 Bdosk mRNA 與蛋白質於黑腹果蠅卵前端。我們成功偵測到 BdOsk 的異位表現，然還未進一步觀察是否可誘導異位生殖細胞於黑腹果蠅胚胎前端產生。儘管如此，我們的研究為 Bdosk 於東方果實蠅卵發育表現的研究奠定基礎，並貢獻於雙翅目昆蟲 oskar 基因演化角色之探討。
並列摘要
Germ cells, by definition, are the cells that can transmit genetic information from generation to generation. In some animals such as the fruit fly Drosophila melanogaster, germ cells are specified at the beginning of development, relying on a specialized cytoplasm called germ plasm. Maternally-inherited germline determinants restricted within the germ plasm are then recruited to the germ (pole) cells that are formed in the posteriormost region of the embryo after fertilization. Among all maternal germline determinants in Drosophila, oskar (osk) mRNA is known to act as molecular anchors to recruit other components of the germ plasm. Posterior localization of osk mRNA in oocytes and early eggs have also been identified in several other holometabolous insects such as mosquitos and wasps, suggesting that restriction of osk transcripts in the developing oocytes labels the forming germ plasm. Based upon this assumption, I cloned an osk ortholog (Bdosk) in oriental fruit fly Bactrocera dorsalis, detecting its expression during oogenesis. Data of in situ hybridization showed that posterior localization of Bdosk mRNA, like that of Drosophila osk, was identified in the posterior poles of the developing oocytes. Localization pattern of the BdOsk protein resembled to that of Bdosk mRNA, indicating that osk plays a conserved role to assemble the germ plasm in the Diptera. In order to verify to which extent the role as a molecular anchor is conserved, we ectopically expressed Bdosk mRNA and protein to the anterior pole of Drosophila oocytes with the aid of the 3’UTR of bicoid (bcd), a noncoding region known to direct bcd mRNA to the oocyte anterior. Anterior localization of BdOsk has been identified but we have not been able to observe whether Bdosk can induce the formation of pole cells in the anterior region of the Drosophila embryos. Notwithstanding, our study paves a path for further investigation of the evolutionary role of osk in the Diptera.