臺灣博碩士論文加值系統

在物種形成的過程中，雜交不合（包括雜交不活與雜交不孕）能夠形成生殖屏障。因此，了解造成雜交不合的基因，將有助於認識物種的起源。OdsH是由unc-4基因複製而產生的一個基因。在秀麗隱桿線蟲中，unc-4與神經發育有關。OdsH是造成兩近緣種果蠅——擬黃果蠅與模里西斯果蠅——雜交子代雄性不孕的主要因子，據信可能是因OdsHmau在精巢中異常表現、或因OdsHmau對擬黃果蠅的Y染色體異常結合所導致。在黃果蠅中，剔除OdsH使雄蟲生殖力下降；免疫染色實驗顯示，缺少OdsH表現的精巢，其生殖細胞數目較少。在本研究中，我測試了六株OdsH RNAi果蠅，其中四株來自果蠅品系中心，兩株為自行建立。我並使用了在特定位置表現綠色螢光蛋白的果蠅，標記精巢內部不同細胞，觀察OdsH對黃果蠅精子生成的影響。本研究展示六株OdsH RNAi果蠅的效果，有助於未來研究OdsH與unc-4的功能重疊。對黃果蠅精巢的觀察結果，則暗示OdsH可能參與精子生成當中的減數分裂與後期分化。

Hybrid incompatibility, including hybrid inviability and hybrid sterility, can act as a reproductive barrier in the process of speciation. Therefore, genes involved in hybrid incompatibility may provide a window on how new biological species form. The gene Odysseus-site homeobox (OdsH) is duplicated from unc-4, a homolog of a neuron developmental gene in Caenorhabditis elegans. OdsH has been identified as a crucial element that triggers hybrid male sterility in reciprocal crosses between two sibling species, Drosophila simulans and D. mauritiana. Previous studies have suggested that misexpression of OdsHmau in testes, or additional binding of OdsHmau to the Y chromosome of D. simulans, results in the hybrid sterility. In D. melanogaster, targeted gene knockout revealed that lack of OdsH expression reduces male fertility. Immunostaining in OdsH null mutant (OdsH0) testes has shown a decrease in the number of germ cells. In this thesis, I generated two OdsH RNAi constructs and examined their efficiency with the other four available at stock centers. In addition, I used reporter lines that express enhanced green fluorescent protein to further characterize this testis-specific gene at the cellular level. My results demonstrated the efficiency of these RNAi strains, which should facilitate future studies on the functional redundancy between OdsH and unc-4. Cytological observation implies that OdsH may play a role in meiosis and terminal differentiation during spermatogenesis.

口試委員會審定書 i
Acknowledgements ii
摘要 iv
Abstract v
Contents vi
List of Figures viii
List of Tables x
Introduction 1
Materials and Methods 7
Fly strains and husbandry 7
Synthesis of mir-6-1-based microRNA for RNAi constructs 7
Generation of RNAi strains against OdsHsim and OdsHmau in D. melanogaster 12
Immunostaining 12
Imaging and quantification 13
Statistical methods 13
RNA extraction and cDNA reverse transcription 13
Results 18
Generation of RNAi strains against OdsHsim and OdsHmau in Drosophila melanogaster 18
RNAi knockdown rescues the OdsHmel ectopic expression phenotype in Drosophila eye 18
His-GFP expression in testes of OdsH+ and OdsH0 flies 25
Sa-GFP expression in testes of OdsH+ and OdsH0 flies 26
BamP-GFP expression in testes of OdsH+ and OdsH0 flies 26
Discussion 32
OdsH RNAi strains should facilitate future studies on the functional divergence of duplicated genes and the normal function of OdsHsim 32
His-GFP expression implies GSC loss or disruption of subsequent spermatogenic development in young OdsH0 flies 35
Sa-GFP expression hints a possible role of OdsH in meiosis or spermatid differentiation 38
BamP-GFP expression yields no information on OdsH in early spermatogenesis 40
References 42
Appendix I: RNAi constructs for transgenesis in Drosophila simulans 45
Generation of pCaSpeR4-based RNAi vectors for P-element transformation 45
Generation of attB-P[acman]-based RNAi vectors for P-element transformation by recombineering 45
Appendix II: mir-6-1-based RNAi design 51

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