vasa-like基因在孤雌生殖豌豆蚜發育過程之表現解析

Abstract

在大部分的動物中，胚胎發育早期生殖細胞即會與體細胞分離出來，以產生配子細胞。生殖細胞如何特化，它們如何移動至性腺，在模式昆蟲黃果蠅 (Drosophila melanogaster) 的研究最為詳盡。在果蠅中，初始生殖細胞 (極細胞) 的形成，依賴聚集於卵後端的生殖細胞決定物質，在多核單細胞之囊胚層形成前，包覆初始生殖細胞的細胞膜將生殖細胞決定物質納入細胞內，使得此類細胞具備發育成生殖細胞之潛力。不同於果蠅模式的非洲沙漠飛蝗 (Schistocerca gregaria) 的生殖細胞特化首度發現是於胚胎發育中期的腹部邊緣細胞。目前尚未發現蝗蟲的卵與早期胚胎有生殖細胞決定物質聚集的現象，因此其生殖細胞的特化較可能藉由鄰近體細胞之訊息分子誘發形成。選用孤雌生殖之豌豆蚜 (Acyrthosiphon pisum) 作為研究生殖細胞特化之材料，原因在於它早期形成囊胚層之模式與果蠅相似，然而其體節發育的方式卻類似於非洲沙漠飛蝗，因此研究此種昆蟲有助於明瞭不同發育形式的物種中，生殖細胞的特化是受到胚胎發育的那個階段所影響。研究策略的初步為先選殖豌豆蚜 vasa 同源基因，vasa 已被發現表現於多種無脊椎與脊椎動物的生殖細胞內，故推測它很有可能也是蚜蟲生殖細胞的標記基因，可作為追蹤生殖細胞的分子標記。本論文已完成兩種豌豆蚜 vasa 基因 (pavasa1 和 pavasa2) 之蛋白質表現與純化，並分別產生此兩種 Vasa 之抗血清。然而，不論是原位雜合或是免疫染色的結果都顯示 pavasa1 和 pavasa2的 mRNA 與蛋白質並非專一性地表現在已可辨識的生殖細胞當中。幸運地，還選殖到第 3 個豌豆蚜 vasa 同源基因 (pavasa3)，並且偵測到它的 mRNA 專一地表現在生殖細胞中，充分顯示pavasa3 為豌豆蚜之生殖細胞標記基因。

Germ cells are the cells that produce gametes, and they are segregated from somatic cells during early development in most animals. In the model insect Drosophila melanogaster, the specification of primordial germ cells (pole cells) depends on the maternal germline determinants localized to the egg posterior. By the formation of syncytial blastoderm, germline determinants are incorporated into pole cells with the forming cell membranes and then these cells are set to become germ cells. Unlike Drosophila, a maternal germ plasm is not identifiable in the grasshopper Schistocerca gregaria and segregated germ cells are first identified in the margin of abdomen undergoing segmentation, suggesting that germline specification may depend on induction signals released from neighboring somatic cells. I selected to study the parthenogenetic pea aphid Acyrthosiphon pisum because of its special pattern of embryogenesis - the formation of blastoderm resembles to Drosophila whilst the adding of segments in later development is similar to Schistocerca. It thus becomes an excellent model for investigating how germline specification is affected by different types of embryogenesis. Homologues of the Drosophila vasa gene are used as germline markers for this study because vasa is highly conserved in germ cells across many species across invertebrates and vertebrates. I expressed and purified fusion proteins of two pea aphid vasa homologues, pavasa1 and pavasa2, using them as antigens to induce anti-Vasa antiserum respectively. Nevertheless, immuno-staining results showed that no localized Vasa signals were identified in the germ cells. Likewise, in situ signals of localized pavasa1 and pavasa2 mRNAs were not germline positive. Fortunately I cloned pavasa3, the third vasa homologue in pea aphids, demonstrating that it was specifically expressed in germ cells. It strongly suggests that pavasa3 is a germline marker in pea aphids and it can be used as a molecular tool for studying germline development in this species.