vasa基因在竹莖扁蚜及豌豆蚜的純化及定性

Abstract

大部份動物的初始生殖細胞，在早期的胚胎發育時期，即自體細胞當中分離。這個現象的發現，在於科學家們觀察到生殖細胞特有之電子的緻密物質nuage，會在核膜之周圍聚集。在很多物種中vasa 或其基因的產物是nuage 的成員之一，這意謂vasa 是一個高度保守的生殖細胞標記。我們從竹莖扁蚜(Pseudoregma bambucocla )及豌豆蚜(Acyrthosiphon pisum )中選殖出vasa -like 基因。其中bsavasa 基因被用來作為分辨竹莖扁蚜的兵蚜及非兵蚜若蟲的指標，因為我們假設在不孕的兵蚜中bsavasa 基因的表現可能會少於非兵蚜。在豌豆蚜中，我們想研究pavasa1 基因在卵生成(oogenesis)及胚胎形成(embryogenesis)過程中如何表現，進而瞭解生殖細胞在這兩個過程中是如何發育的。由反轉錄聚合酵素連鎖反應(RT-PCR)的結果中顯示pavasa1 及bsavasa 皆表現於成蟲的卵巢，顯示bsavasa 及pavasa1 可能為生殖細胞的標記基因。然而在竹莖扁蚜的兵的表現被偵測到。我們推測bsavasa 在一齡兵蚜的未退化生殖細胞當中表現，或者bsavasa 在其體細胞亦有表現，因此RT-PCR 出現“bsavasa -positive ”的結果。我們利用北方雜合法(Northern Blot)辨識出bsavasa及pavasa1 的mRNA 約為3 kb，推測其二者長度大約相同。我們並沒有成功的擴增由D.Stern 提供的pavasa1 3 ’端的序列，可能是有大量的poly-A 序列在其3 ′端，消耗掉大量用於3 ′ RACE-PCR 的poly-T 引子。為了知道pavasa1 是否為單(single-copy)基因，我們將豌豆蚜的基因 (genomic DNA)用不同的限制酵素(restriction enzyme)作切割，並用DIG 標記的vasa DNA 作為探針(probe)。結果顯示單一個限制酵素切割後的基因體DNA 只有單一條帶 (single band)被偵測到(HindIII:~6 kb;SpeI:~7kb;EcoRI:~3.5 kb)。由此結果我們可推測pavasa1 是單一基因。比對Pavasa1 及本實驗室選殖的Pavasa2 蛋白質序列，只有38 ﹪相同，並且在N-端之序列呈現分歧，顯示在豌豆蚜基因體中至少有兩個不同的vasa 基因。本文的最後一部分，我們將根據本實驗室所提供的pavasa1 原位雜合結果與抗體染色的數據來討論pavasa1 是否為生殖細胞之標記基因。

Primordial germ cells (PGC) are segregated from somatic cells during early embryogenesis in most animals. The phenomenon is known by the observation of “nuage”, a group of electron-dense materials in the periphery of nuclear membrane. Vasa gene or its product has been known as a component of nuage in many species, showing that it is a conserved germline marker. In our study we cloned vasa-like genes in the bamboo stem aphid Pseudoregma bambucicola (bsavasa) and in the pea aphid Acyrthosiphon pisum (pavasa1). The bsavasa was used to discriminate the soldier and non-soldier nymphs, on the hypothesis that the expression of bsavasa is weaker in sterile soldiers; pavasa1 was used as a marker to study germline development in parthenogenetic aphids because we wanted to know its developmental expression from oogenesis to embryogenesis within an ovariole. RT-PCR shows that vasa mRNA was preferentially identified in the adult ovary, suggesting that it is a potential PGC marker in these two aphid species. However, expression of vasa was detected in soldier and non-soldier nymphs of bamboo stem aphids, implying that either bsavasa is expressed in soldiers’ non-degenerated germ cells or it is also expressed in somatic tissues at the 1st instar nymph. We identified a ~3 kb transcript of vasa with Northern blot in both species of aphids, implying that the length of vasa mRNA may be similar. Our effort to clone the 3’-end sequence of pavasa1 did not work, which was similar to the sequence provided by D. Stern (Princeton, USA). It might be caused by the abundant poly-A sequences scattering at the 3’-end area within the open reading frame, which consumed most of the poly-T primers in the 3’ RACE-PCR. In order to investigate whether pavasa1 is a single-copy gene, blots containing genomes digested respectively with HindIII, SpeI and EcoRI were probed with DIG-labeled vasa genes. Repeated results show that a single band was identified in each lane from different enzyme digestion (HindIII: ∼6kb; SpeI: ∼7 kb; EcoRI: ∼3.5 kb), suggesting that this vasa gene (pavasa1) is single copy. We also compared the deduced protein sequence encoded by pavasa1 with that encoded by pavasa2, a second vasa-like gene cloned in our laboratory. Sequence alignment shows that these two Vasa proteins only have 38% identity and N-terminal sequences appear divergent. It shows that there are at least two different vasa genes existing in the pea-aphid genome. In the last part, we will discuss whether pavasa1 is a germline marker according to the provided results of in situ hybridization and antibody staining.