李後晶臺灣大學：昆蟲學研究所呂曉鈴Lu, Shau-LinShau-LinLu2007-11-262018-06-292007-11-262018-06-292005http://ntur.lib.ntu.edu.tw//handle/246246/55024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.I. Introduction A. General background and Research Aims 1 B. Modes of Germline Development 2 C. Vasa Gene and Germline Development 4 II. Materials and Methods A. Insects 6 B. Total RNA preparation 6 C. Genomic DNA preparation 6 D. Plasmid DNA preparation 7 E. Reverse Transcription 7 F. Polymerase Chain Reaction 8 G. Subcloning 8 H. RACE PCR 9 I. DIG-Labeled DNA Probe Synthesis 10 J. Northern Blot 11 K. Southern Blot 13 III. Results A. Cloning and Detection of The Expression of Vasa mRNA in The Bamboo Stem Aphids, Pseudoregma bambucicola 16 B. RACE PCR Cloning and Detection of The Expression of Vasa mRNA in The Pea aphids, Acyrthosiphon pisum 17 C. Identification The Size of Vasa Transcripts with Northern Blotting 18 D. Examination of Gene Duplication with Southern Blotting 19 IV. Discussion 20 V. References 22 VI. Appendix A. The Basic Concept of Aphid Biology 31 B. The Bamboo Stem Aphids – Pseudoregma bambucicola 33 C. The Embryogenesis of Aphids 35 Table List Table 1 The Bsavasa protein sequence blast analysis 38 Table 2 The Pavasa1 protein sequence blast analysis 39 Table 3 The bamboo stem aphid soldier contrast list between primary and secondary host 40 Table 4 Sequence identity (%) between four DEAD-box proteins: Bsavasa, Pavasa1, eIF4A and Rm62 40 Figure List Fig. 1A Partial sequences of bsavasa and its gene product 41 Fig. 1B Detection of the expression of bsavasa mRNA with RT-PCR 42 Fig. 2 Detection of the expression of bsavasa mRNA with RT-PCR 43 Fig. 3 Detection of the expression of pavasa1 mRNA with RT-PCR 44 Fig. 4 Comparison of Pavasa1 and other insect Vasa protein sequences 45 Fig. 5 Comparison of Pavasa1, Drosophila Vasa and Bombyx mori Vasa protein sequences 46 Fig. 6 Strategies for 3’RACE PCR to clone 3’end sequences of pavasa1 gene 47 Fig. 7 Plasmid map of pavasa1 F864-R1439 in the pGEM-T easy vector 48 Fig. 8 PCR-synthesis of the DIG-labeled DNA probes 49 Fig. 9 Northern blots containing total RNA probed with pavasa1 (F864-R1439) DIG-labeled DNA 50 Fig. 10 Northern blots containing total RNA probed with pavasa1 (F484-R1439) DIG-labeled DNA 51 Fig. 11 Northern blot for the detection of the length of the bamboo stem aphid vasa (bsavasa) transcript 52 Fig. 12 Southern blotting results 53 Fig. 13 Comparison of Pavasa1 and Pavasa2 protein sequences 54 Fig. 14 Southern blots hybridized with DIG-labeled pavasa1 and pavasa2 probes 55 Fig. 15 Expression of pavasa1 mRNA in the germarium, oocyte and embryos 56 Fig. 16 The pea aphid’s ovary staining with Formosa 2 antibody 57 Fig. 17 The life cycle of Pseudoregma bambucicola in Taiwan 58en-US竹莖扁蚜豌豆蚜vasaaphidsthesis