黃果蠅初期種化機制之行為及遺傳分析｜國立清華大學博碩士論文庫

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研究生：	郭崇涵 Tsung-Han Kuo
論文名稱：	黃果蠅初期種化機制之行為及遺傳分析 Behavioral and genetic studies of incipient speciation in Drosophila melanogaster
指導教授：	丁照棣 Chau-Ti Ting 方淑 Shu Fang
口試委員:
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 分子與細胞生物研究所 Institute of Molecular and Cellular Biology
論文出版年：	2004
畢業學年度：	92
語文別：	英文
論文頁數：	80
中文關鍵詞：	果蠅、交尾前生殖隔離、交配行為、表皮碳氫化合物、辛巴威、求偶
外文關鍵詞：	fly, pre-mating isolation, mating behavior, cuticular hydrocarbon, Zimbabwe, courtship
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種化的發生對於生物學家一直是一個有趣但難解的問題。交配前生殖隔離在自然族群的種化形成具有強大的推動力，但其詳細機制不易在實驗室中探討。黃果蠅在不同族群中交配喜好的差異提供了良好的機會去探索初期種化的機制。辛巴威(Z型)的雌果蠅在有選擇的情況下只會和辛巴威的雄果蠅交尾，而其他族群(M型)的雌果蠅則不具有明顯分辨的能力。這種行為系統同時也引發一系列相關的議題，譬如其它的隔離機制是否存在，造成此行為差異的遺傳基礎為何，以及雌果蠅所辨識的特徵，都是我們極欲回答的問題。首先，為了找出維持這種行為上多型性的原因，我利用各種不同的行為實驗找出M型雌果蠅在接受求偶行為上的偏好性，並發現Z型雄果蠅在求偶時有比較積極的行為。在雄果蠅求偶順序圖中也可見到這種交尾隔離並不是單方向的。其次，藉由確認兩型果蠅身上帶有不同表皮碳氫化合物以及氣味轉移實驗，雌果蠅被證實會藉由雄果蠅的表皮碳氫化合物來辨識雄果蠅。最後，為了尋找這初期種化機制相關的基因，我們利用減除式雜交法和基因微陣列實驗，找到了許多表現有差異的基因，這些基因很有可能是種化基因值得我們作更深入的研究。透過各種不同的實驗設計，我們可以得到更多和這個交尾前生殖隔離相關的知識和訊息。

How speciation occurred has always been an intriguing and mysterious issue to evolutionary biologists. Pre-mating isolation exhibits an evolutionary force to prevent gene exchange in natural populations but the detailed mechanism is difficult to study in the laboratory. Drosophila melanogaster provides an opportunity to examine an incipient speciation process because of the divergent mating preference between different races. Zimbabwe females, under multiple-choice conditions, mate only with males from the same race whereas elsewhere females show weak preference. This behavior system raises several interesting issues such as the existence of other isolation mechanism, the underlying genetics of this sexual isolation system, and the precise traits for female recognition. In this study, first, I would like to know if other factors maintain this sexual isolation. M female preference and the aggressiveness of Z male courtship were identified via different behavioral assays. Analyses of male courtship also showed that the mating isolation is bi-directional. Second, through comparison of the cuticular hydrocarbon profiles between Z and M populations and perfuming assays, males’ cuticular hydrocarbon profiles are first identified traits responsible for Z females’ recognition. Finally, in order to find the genes related to this incipient speciation, several differentially expressed genes were identified using subtractive hybridization and cDNA microarrray experiments. All of these genes are candidates as speciation genes and worthy for further study. Through different experiment designs, we could obtain more information about this pre-mating isolation system.

Contents

Chinese Abstract………………………………………………………………….…………...i
Abstract………………………………………………………………….………….................ii
Acknowledgements..…………………………………………………….…………...............iii
Chapter 1 General Introduction……………………………………………….……...……..1
Chapter 2 Dissection of Mating Behavior under the Sexual Isolation in Drosophila melanogaster….………………………………………………………………….…………....5
    Introduction……………………………………………………………………………...5
    Materials and Methods……………………………………………….…………………8
    Results………………………………………………..…………………........................13
    Discussion……………………………………………….………………………………26
Chapter 3 Pheromonal Cuticular Hydrocarbons Affecting Sexual Isolation in Drosophila melanogaster….………………………………………………………………….…………..29
    Introduction…………………………………………………………………………….29
    Materials and Methods………………………………………………….……………..32
    Results………………………………………………..…………………........................34
    Discussion……………………………………………….………………………...……44
Chapter 4 Search for Differentially Expressed Gene Involved in the Sexual Isolation in Drosophila melanogaster……………………………………………..……………..………46
    Introduction……………………………………………………………………………46
    Materials and Methods………………………………………………….…………….49
    Results………………………………………………..………………….......................59
    Discussion……………………………………………….……………………………...72
References.……………………………………………………………………….…….….....76
List of Tables

Table 2.1 Mating patterns between D. melanogaster Zimbabwe (Z) and Cosmopolitan (M) races in multiple-choice experiments………………………..……………………14
Table 2.2 Female choice between males of the Zimbabwe (Z) and cosmopolitan (M) races of D. melanogaster…………………………………………………………………..16
Table 2.3 Mating parameters (mean ± standard error) of individual pairs of flies from Z (Z30) and M (FR) strains……………………………………….……………………….18
Table 2.4 Male choice between females of the Zimbabwe (Z) and cosmopolitan (M) races of D. melanogaster…………………………………………………………………..21
Table 2.5 The proportion of male courtship behaviors (% mean ± standard error) of individual pairs of flies from Z (Z30) and M (FR) strains…………………...…...24
Table 3.1 Composition of male cuticular hydrocarbons (% mean ± standard error) in different populations of Drosophila melanogaster…………………………………....……36
Table 3.2 Percentages of male cuticular hydrocarbons (% mean ± standard error) of pure strains used in perfuming experiments and ‘target’ flies crowded with ‘donor’ flies………………………………………………………………………………..39
Table 3.3 Percentages of male cuticular hydrocarbons (% mean ± standard error) of different whole-chromosome substitution lines.………………………...……….…………43
Table 4.1 Primers designed for real-time PCR……..……………………………………...…57
Table 4.2 Identification of the female candidate clones selected by the subtractive hybridization method..…………………………………………………………………....…...…62
Table 4.3 Identification of the male candidate clones selected by the subtractive hybridization method……………………….……………………………………………………63
Table 4.4 Candidate male genes selected from the microarray result with the ratio of expression level for Z males relative to M males larger than 1.49…...…….....….65

Table 4.5 Candidate male genes selected from the microarray result with the ratio of expression level for M males relative to Z males larger than 1.49………...……..66
Table 4.6 Candidate female genes selected from the microarray result with the ratio of expression level for Z females relative to M females larger than 1.49...…………67
Table 4.7 Candidate female genes selected from the microarray result with the ratio of expression level for M females relative to Z females larger than 1.49….………..68
List of Figures

Figure 2.1 Illustration of male courtship parameters…………………………...……11
Figure 2.2 Graphs of behavior transition of male courtship…………………………23
Figure 3.1 Typical gas chromatograms of a male M (FR) and Z (ZS11) of Drosophila melanogaster………………..……………………………………………35
Figure 3.2 Comparison of mating pairs……………………...………………………41
Figure 4.1 The procedure of Subtractive hybridization protocol……………..……..52
Figure 4.2 PCR products of the subtractive hybridization………………..…………59
Figure 4.3 Verification of insertion by PCR with M13 forward and reverse primers…………………...……………………………….………………60
Figure 4.4 One of dot blot results representing the screening condition…………….61
Figure 4.5 Numbers of candidate genes selected from cDNA microarray experiments…..………………………………………………….….……64
Figure 4.6 Quantification of the expression levels of candidate genes from the female FR subtracted library.………………………………………………….…71
Figure 4.7 Quantification of the expression levels of candidate genes from the female MMZ subtracted library…...………………………………….……….…71
Figure 4.8 Quantification of the expression levels of candidate genes from microarrays….…………………………………………………….……72

                                

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