開發螢光偵測技術探討發育基因於孤雌胎生豌豆蚜之非典型表現

Exploration of non-canonical expression of developmental genes in the asexual viviparous pea aphid using fluorescence approaches

鍾成侑(Chen-yo Chung)

指導教授 ： 張俊哲

國立臺灣大學/生物資源暨農學院/昆蟲學研究所/博士(2014年)

https://doi.org/10.6342/NTU.2014.10446

摘要

豌豆蚜為半翅目昆蟲中第一個被全基因體解序的昆蟲，由於其複雜生活史中的非遺傳多型性以及不同型式的胚胎發育使之成為發育生物學中獨特的模式物種，為更進一步瞭解豌豆蚜胚胎發育過程，解開發育基因之表現型式為不可或缺的方法，為達到此一目的，分子工具以及基因表現分析皆同等重要。早先，全體原位雜合反應已被發展並運用於監測基因於豌豆蚜卵巢的表現，但此一技術在同時偵測兩基因表現及建立三維空間表現上有其極限，因此，我發展了可信賴的全體螢光原位雜合程序來解決這些問題，利用不同螢光染色原理的優勢，我成功偵測到基因在不同組織間—如胚外以及胚體—的表現，此外，結合不同方式所進行的雙色螢光原位雜合也成功讓我同時觀察到兩個基因於胚胎中的表現，此螢光原位雜合程序也幫助我在接下來的研究中，解析發育基因的表現。在 2010 年由本實驗室所發表的報告中，我們發現 Aphb 信使核糖核酸 (messenger RNA) 除表現在早期胚胎前端，也會表現在中、晚期體節以及中央神經系統 (CNS) 中，顯示 Aphb 擁有保守的表現位置。在本研究中，我發現 Aphb 有一新的表現在生殖細胞，此生殖細胞的表現最早出現自原始生殖細胞 (primordial germ cell, PGC)，並且持續到胚胎最晚期，甚至也在形成中的生殖原區及其產生的卵母細胞前端表現。這樣的表現顯示Aphb 在早期可能取代了 bicoid 的角色，以及接下來時期可能協助生殖細胞的形成。為推測 Nanos (Nos) 及 Pumilio (Pum) 複合體是否可能抑制後端 Aphb 的轉譯，我分析了 Appum 蛋白質的結構以及信使核糖核酸表現，高度保守的 PUM-HD 顯示 ApPum 能夠抑制 Aphb 的轉譯，雖然沒有偵測到不對稱性的信使核糖核酸表現。將此結果與過去 Aphb, Appum, and ApNos表現做比較，我發現豌豆蚜在後端體軸決定是倚賴 Nos 及 Pum 複合體，但前端則是被 Aphb 決定。

關鍵字

豌豆蚜 ； 前後體軸 ； 全體螢光原位雜合 ； 生殖細胞 ； hunchback ； pumilio

並列摘要

The pea aphid Acyrthosiphon pisum is a genomic model insect and a unique model for polyphenism due to its developmental plasticity in response to environmental cues. To uncover the relation of embryonic development and gene regulations, reliable expression protocols and functional tools are required. Whole-mount in situ hybridization (WISH) we previously reported can be used to monitor gene expressions during embryogenesis, however chromogenic signals are defective in double detection of genes and construction of three-dimensional image. I therefore developed a fluorescent in situ hybridization (FISH) protocol to overcome these defects. By means of different advantages of four FISH methods, I successfully detected gene expressions in somatic and extraembryonic tissues. The combination of FISH methods also allowed the double detection of genes in somatic cells, germ cells, or both in one preparation. This FISH protocol further aids me in revealing the expression of developmental genes. In our previous findings, mRNA expression of A. pisum hunchback (Aphb), a Drosophila homolog of hunchback, was found in the segments and central nervous system of mid/late stages apart from the anterior pole of early stages, implicating its conserved roles among arthropods and lower organisms. Here I discovered a novel expression pattern of Aphb in germ cells of the pea aphid. Germline expression of Aphb initiates while primordial germ cells formed, and maintains throughout developmental stages. In late embryos, Aphb is also expressed in maturing germaria as well as the protruding oocytes. These findings implicate that the homolog of hb in aphids replaces the role of bicoid in anterior determination and, moreover, has the roles in formation of germ cells. To reveal whether the complex of Nanos (Nos) and Pumilio (Pum) is required to repress the translation of anterior-localized Aphb in the posterior, I analyzed the structure of A. pisum Pum (ApPum) protein and the expression patterns of Appum mRNA. The highly conserved protein structure indicates the ApPum can repress the translation of Aphb, though the asymmetric expression of Appum mRNA, like Drosophila pum, was not found. Together with the known expression patterns of Aphb, Appum, and A. pisum Nos (ApNos), it appears that posterior determination of the pea aphid relies on the ApNos/ApPum complex and the anterior is determined by Aphb.

並列關鍵字

Acyrthosiphon pisum ； anteroposterior axis ； whole-mount fluorescent in situ hybridization (FISH) ； germ cells ； hunchback ； pumilio

參考文獻

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