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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/16117完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 張俊哲(Chun-che Chang) | |
| dc.contributor.author | Gee-way Lin | en |
| dc.contributor.author | 林季瑋 | zh_TW |
| dc.date.accessioned | 2021-06-07T18:01:38Z | - |
| dc.date.copyright | 2012-08-28 | |
| dc.date.issued | 2012 | |
| dc.date.submitted | 2012-08-03 | |
| dc.identifier.citation | Abdelhaleem, M. (2005). RNA helicases: regulators of differentiation. Clin Biochem 38, 499-503.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/16117 | - |
| dc.description.abstract | 在昆蟲中，生殖細胞在胚胎發育的過程中與體細胞分離，是生殖細胞特化相當重要的一步。部分的物種如黃果蠅，生殖細胞在發育早期即與體細胞分離。黃果蠅胚後端的核在細胞化時，囊括了聚集於胚後端的生殖細胞決定物質，而被特化成為生殖前驅細胞；其他的物種如蝗蟲，生殖前驅細胞在原腸胚形成後才與體細胞分離，且推測生殖細胞的特化是透過其鄰近的體細胞釋放出訊息誘導而成。上述生殖細胞的研究成果，都是透過偵測 vasa 基因的表現，因為 vasa 基因保守表現於各種動物的生殖細胞中，成為最好的生殖細胞標記基因。
本論文的研究當中，我選殖出豌豆蚜的四個 vasa 同源基因，製備分別辨識其蛋白的四個抗體，偵測四個 vasa 基因訊息 RNA 以及蛋白在發育過程中的表現。旨在探討無性和有性的豌豆蚜胚胎，其生殖細胞如何特化。以及同一基因體在不同光週期的刺激之下，能產生無性胚胎或有性胚胎的可塑性，是否受到四個 vasa 基因差異性的表現來調控。 研究結果顯示，無性胚胎和有性胚胎都是藉由同一個 Vasa 蛋白來特化其生殖細胞。不過，無性胚胎中，Vasa 蛋白聚集於胚後端，並會在生殖細胞特化之前向前端位移。Vasa 蛋白聚集的位移可能是現今無性且胎生的蚜蟲才出現的特徵。這個假設需透過分析較原始的蚜蟲物種，其具有無性生殖且卵生的世代才能驗證。除此之外，蚜蟲 Vasa 蛋白在胚後端聚集的分子機制是否和其它昆蟲有共通性，以及蚜蟲四個 vasa 基因的功能性分析，都需要繼續探討。 | zh_TW |
| dc.description.abstract | Segregation of germline from soma during embryogenesis is critical to the specification of germ cells in insects. In some species such as the fruit fly Drosophila melanogaster, germ cells are segregated during early development. It has been reported that in D. melanogaster the posterior syncytial nuclei are associated with the maternally inherited germline determinants in the egg posterior to form the primordial germ cells (PGCs) at the beginning of embryogenesis. In some other species like the grasshopper Schistocerca gregaria, segregation of PGCs is first identified after gastrulation and germline specification may be achieved via signal induction from neighboring somatic cells. Discoveries of germline segregation mentioned above were all evidenced by the expression of vasa, a highly conserved germline marker in insects and other animals.
In this study, I cloned four vasa genes (Apvasa1-4) in the pea aphid Acyrthosiphon pisum, made antibodies against ApVasa1-4, and detected the developmental expressions of Apvasa mRNAs and ApVasa proteins. My ultimate goals are to understand how germline is specified in asexual and sexual pea aphids and whether reproductive plasticity of the pea aphid is regulated by differential expressions of four vasa genes from the same genome. My results show that only one germline specific Vasa protein to specify PGCs in asexual and sexual embryos. The localization of germline specific Vasa protein is dymanic in asexual embryo, in which accumulated Vasa protein shifts its position toward anterior of embryo. I suggest that the dynamic localization of Vasa protein in asexual embryo is a derived feature in modern aphids, which are viviparous in asexual phase. This hypothesis has to be confirmed by analyzing the localization of Vasa protein in ancient asexual and oviparous aphids. Besides, the conserved mechanism for the localization of germline specifc Vasa in aphids across other insects, and the functional assay of four vasa genes are also necessary to be done. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-07T18:01:38Z (GMT). No. of bitstreams: 1 ntu-101-D95632002-1.pdf: 42573414 bytes, checksum: 234cdd7252f174f0c701c8745d3f7ac9 (MD5) Previous issue date: 2012 | en |
| dc.description.tableofcontents | 中文摘要.................................................................................................... i
Abstract.................................................................................................... ii Abbreviations............................................................................................ iv Table of contents...................................................................................... vii Chapter 1. General introduction 1.1 Germline development 1 Strategies of germline segregation 1 Modes of germline specification 2 1.2 Germline marker genes, vasa and nanos 3 Vasa is a member of DEAD-box protein family 4 Functions of Vasa protein in germline 5 The zinc-finger protein, Nanos 7 1.3 Characterization of germline specification mode with expression of vasa in insects 7 1.4 Biology of aphid 10 The annul life cycle of aphids 10 Developmental plasticity of aphids 11 Evolution of reproductive phases in aphids 12 The strians of the pea aphid in this study 13 1.5 Specific aims 14 Chapter 2. Germline development in the asexual embryos of viviparous pea aphid 2.1 Background 15 The molecular network for germ plasm assembly 16 Embryonic development of asexual embryos in the viviparous aphids 16 Germline development in the asexual embryos of viviparous aphids 17 Germline specification in the asexual embryos of viviparous pea aphid 18 Germline migration in model organisms 18 2.2 Materials and methods 19 The asexual pea aphid culture 19 Cloning of the pea aphid vasa (Apvasa1) and nanos (Apnanos) genes 19 Synthesis of Apvasa1 and Apnanos in situ riboprobes 21 Whole-mount in situ hybridization in the asexual embryos 22 2.3 Results 23 Probe accessibility of whole-mount in situ hybridization 25 Apvasa1 RNA probes traced the germline migration in the asexual embryos 25 Apnanos marked the presumptive primodium germ cells and germ cells in the gonads 26 2.4 Discussion 28 Figures in Chapter 2 29 Figure 2.1 Whole-mount in situ of Apvasa1 on the early asexual embryos 30 Figure 2.2 Whole-mount in situ of Apvasa1 on asexual embryos after stage 7 onward 31 Chapter 3. Differential expressions of four vasa homologs Apvasa1 to Apvasa4 during asexual embryogenesis 3.1 Background 32 3.2 Materials and methods 33 The asexual pea aphid culture 33 Cloning of the other pea aphid vasa (Apvasa2 to 4) genes 33 Making of anti-ApVasa1 to 4 antibodies 33 Western blot analysis 34 Whole-mount immunolabeling of the asexual embryos 34 3.3 Results 35 Molecular characterization of vasa homologues in the pea aphid 35 Expression of Apvasa1 to 4 RNA during asexual embryogenesis 36 Dynamic localization of ApVasa1 protein in the posterior to specify germline during early asexual embryogenesis 38 Specified primodium germ cells were marked with ApVasa1 antibody during asexual embryogenesis 40 Other three ApVasa proteins were not germline specific in the asexual embryos 40 ApVasa1 antibody cross-reacted with the green peach aphid Myzus persica Vasa protein in germline 41 3.4 Discussion 42 Figures and Tables in Chapter 3 44 Figure 3.1 Alignments of the pea aphid Vasa protein sequences 45 Figure 3.2 Schematic comparisons between the pea aphid Vasa proteins, insect Vasa proteins, and the pea aphid non-Vasa DEAD-box proteins 47 Figure 3.3 Whole-mount in situ hybridization of Apvasa1 to 4 on asexual early stage of embryos (stage 0 to 5) 48 Figure 3.4 Whole-mount in situ hybridization of Apvasa1 to 4 on asexual early stage of embryos (stage 6 to 11) 50 Figure 3.5 Whole-mount in situ hybridization of Apvasa1 to 4 on asexual mid stage of embryos (stage 11 to 14) 51 Figure 3.6 Whole-mount in situ hybridization of Apvasa1 to 4 on asexual late stage of embryos (stage 15 to 19) 53 Figure 3.7 Increasing intensities of Apvasa3 transcripts in non-germline cells on asexual early to mid stages of embryos (stage 5 to 10) 56 Figure 3.8 Accumulation of ApVasa1 protein in the posterior of oocytes in the asexual aphids (stage 0 to 1) 58 Figure 3.9 Accumulation of ApVasa1 protein in the posterior of oocytes in the asexual aphids (stage 1 to 2) 59 Figure 3.10 Accumulation of ApVasa1 protein in the posterior of asexual embryos (stage 3) 60 Figure 3.11 Dynamic posterior localization of ApVasa1 protein in the asexual embryos (stage 4) 62 Figure 3.12 Presumptive PGCs are specified by recruitment of posteriorly localized ApVasa1 protein in the asexual embryos (stage 5 to 6) 64 Figure 3.13 Germline development is traced by ApVasa1 antibody during asexual embryogenesis (stage 7 to 19) 66 Figure 3.14 Improvement of whole-mount immunostaining on elder stages of asexual embryos 68 Figure 3.15 Migrating germline is stably detected by ApVasa1 antibody in the all stages of asexual embryos after proteinase K treatment (stage 9 to 19) 69 Figure 3.16 Developmental expressions of ApVasa2 to 4 antibodies during asexual embryogenesis (stage 0 to 12) 70 Figure 3.17 ApVasa1 antibody cross-reacted with the green peach aphid Myzus persica Vasa protein in germline during asexual embryogenesis 71 Figure 3.18 Western blot of ApVasa1 to 4 fusion proteins with ApVasa1 to 4 antibodies 72 Figure 3.19 Western blot of the pea aphid ovary proteins in the asexual phase with ApVasa1 to 4 antibodies 73 Table 3.1 Primers for RACE PCR 74 Table 3.2 Primers for making in situ of Apvasa1 to 4 constructs 74 Table 3.3 Primers for cloning ApVasa1 to 4 fusion proteins as antigens 74 Chapter 4. Germline development in the sexual eggs of the pea aphid 4.1 Background 75 Embryogenesis within the sexual eggs of the pea aphid 75 Germline development during embryogenesis within the sexual eggs of the pea aphid 77 4.2 Materials and methods 77 Induction for sexual phase of the pea aphid 77 Fixation, dissection and immunostaining of the sexual eggs 78 RNA extraction and real-time quantitative PCR 80 Western blot analysis 81 4.3 Results 81 Maternal expressions of Apvasa1 ot 4 during oogenesis in the sexual females 81 Accumulation of ApVasa1 protein in the posterior of syncytial embryos within the sexual eggs specified their germline 82 Germline development during embryogenesis within the sexual eggs 82 Differential expression of Apvasa1 to 4 in the sexual phase 83 4.4 Discussion 84 Posteriorly localized ApVasa1 and aggregation of ApVasa4 as nuage were conserved in both reproductive phases 84 ApVasa3 was specific assembled on the oocyte nucleus of sexual females 85 The mode of germline specification and the recruitment of ApVasa1 protein were conserved both in asexual and sexual phases 85 Figures in Chapter 4 87 Figure 4.1 Schedule of the induction for sexual phase 88 Figure 4.2 Maternal expressions of ApVasa1 to 4 proteins during oogenesis in the sexual females 89 Figure 4.3 Accumulation of ApVasa1 protein in the posterior of syncytial embryos within sexual eggs 91 Figure 4.4 ApVasa1protein is recruited in the presumptive PGCs within the sexual eggs 92 Figure 4.5 Germline development is closed associated with bacteria during early gastrulation of embryos within the sexual eggs 93 Figure 4.6 Germline development within the sexual eggs after 3 days AEL onward 95 Figure 4.7 Western blots of protein extracts in the sexual phase with ApVasa1 to 4 antibodies 97 Figure 4.8 Quantitative real-time PCR of differential expressions of Apvasa1 to 4 98 Figure 4.9 Quantitative real-time PCR of differential expressions of Apvasa1 to 4 among ovaries from different stages of sexual instars 99 Chapter 5. Discussion 5.1 A comparison of germline development in asexual and sexual phase 100 The posteriorly localized Vasa protein is conserved for germline specification in asexual and sexual phase of the pea aphid 101 Migration of extra-embryonic germline is divergent within the asexual egg chambers and sexual eggs 101 Evolution of Vasa proteins assembly in the presumptive germ plasm within aphids 102 Comparison of germline specification among insects 103 5.2 Expressions and functions of Vasa in non-germline 104 Expression sof Vasa in non-germline 104 Functions of Vasa in multipotent cells 105 Differential expressions of multiple vasa genes 105 5.3 Accessibility of functional assay for vasa genes in aphids 107 The interaction of Vasa with RNAi pathway 108 5.4 Regulation of vasa expression in germline 109 5.5 Future aspects 110 Figure in Chapter 5 111 Figure 5.1 Germline specification in insects 112 References 113 Appendix Appendix 1.1 Strategies of germline segregation 123 Appendix 1.2 Localization of germline specification molecules across the Metazoa 124 Appendix 1.3 Function motifs and protein structure of Vasa 125 Appendix 1.4 Expression of Vasa protein in the locust Schistocerca embryos 126 Appendix 1.5 The annul life cycle of the pea aphid 127 Appendix 2.1 Germ plasm assembly in Drosophila oocyte 128 Appendix 2.2 Embryogenesis of asexual and viviparous aphids 129 Appendix 2.3 Staging scheme of the asexual embryos in the pea aphid 130 Appendix 2.4 Germline specification in the asexual embryos 132 Appendix 2.5 Publication: Whole-mount identification of gene transcripts in aphids: protocals and evaluation probe accessibility. Appendix 2.6 Publication: Apvasa marks germ-cell migration in the parthenogenetic pea aphid Acyrthosiphon pisum (Hemiptera: Aphidoidea) Appendix 2.7 Publication: Developmental expression of Apnanos during oogenesis and embryogenesis in the parthenogebetic pea aphid Acyrthosiphon pisum. Appendix 4.1 Embryonic development of the sexual eggs in the grain aphid T. graminum 133 Appendix 4.2 Embryogenesis within the sexual eggs in the first two weeks of the pea aphid 134 Appendix 4.3 Embryogenesis within the sexual eggs after two weeks of the pea aphid 136 | |
| dc.language.iso | en | |
| dc.title | 由 vasa 基因表現探討豌豆蚜生殖細胞發育之可塑性 | zh_TW |
| dc.title | Developmental plasticity of germline development in the pea aphid Acyrthosiphon pisum: implicated by vasa expressions | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 100-2 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 郭保麟,路光暉,陳俊宏,李士傑,林明德 | |
| dc.subject.keyword | 豌豆蚜,生殖前驅細胞,生殖細胞特化,Vasa,生殖的可塑性,無性胚胎,有性胚胎, | zh_TW |
| dc.subject.keyword | pea aphid,PGCs,germline specification,Vasa,reproductive plasticity,asexual embryo,sexual embryo., | en |
| dc.relation.page | 137 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2012-08-06 | |
| dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
| dc.contributor.author-dept | 昆蟲學研究所 | zh_TW |
| 顯示於系所單位： | 昆蟲學系 | |
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