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研究生中文姓名:鄭顥生
研究生英文姓名:Cheng, Hao-Sheng
中文論文名稱:南美白蝦的生殖調控:性別特異性基因分析與IAG基因對精巢發育的影響
英文論文名稱:Molecular characteristics and expression patterns of IAG and sex-dimorphic genes during gonadal development of pacific white shrimp, Litopenaeus vannamei
指導教授姓名:張清風
吳貫忠
口試委員中文姓名:教授︰李奇英
教授︰岳文勛
教授︰鄭絢如
教授︰張清風
助理教授︰吳貫忠
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:水產養殖學系
學號:10433024
請選擇論文與海洋研究相關度:直接相關
請選擇論文為:學術型
畢業年度:106
畢業學年度:105
學期:
語文別:中文
論文頁數:91
中文關鍵詞:轉錄體雄性腺精巢發育
英文關鍵字:TranscriptomeTestis developmentAndrogenic gland
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南美白蝦為現今全球最重要的經濟性養殖甲殼類物種之一,而包含白蝦在內的所有對蝦物種都具有性別二型性,雌蝦比雄蝦具有更大的體型與更佳的成長率,因此,若能建立全雌化的養殖族群便能使養殖的效益大大提升。為瞭解白蝦性別發育的機制,我們針對主要參與甲殼類性別分化的3個器官,精巢、卵巢與雄性腺進行分析,於本研究主要分為兩個部份,第一部份的實驗以前述3個組織為材料建立轉錄體資料庫,並篩選出具有性別特異性表現的基因,透過qPCR與原位雜交染色分析這些基因的表現模式;第二部份則自白蝦雄性腺樣本中選殖出了甲殼類雄性性別分化的調控因子insulin-like androgenic gland hormone(IAG),分析其在組織分佈、蝦苗發育階段上的表現模式,並透過摘除雄性腺及摘除眼柄的實驗探討眼柄與雄性腺對白蝦精巢發育的影響。於第一部份的實驗中篩選出的具有雄性特異性的基因包括DMC1和haywire-like(參與減數分裂的分子標記)、MIPP1、MIPP2及glass-like(可能參與雄性性別發育),具有卵巢特異性表現的基因包括OPTP、PTP和PTPI(卵細胞中皮質桿的結構蛋白)、TSP、TSPIIA與TSPIIB(可能調控卵巢發育)、nudeI(果蠅濾泡細胞專一表現基因)以及contig223(初級卵母細胞大量表現的新基因)。而除了上述基因外,亦從轉錄體中鑑定出了其他可能參與性別分化的基因,包括TRA2、FOXL2、SXL,其中TRA2與SXL在精卵巢間表現量無顯著差異,而FOXL2則在精巢中有較高表現。第二部份的實驗結果顯示白蝦IAG的表現不具有雄性腺專一性,在包含雄性腺的輸精管、眼柄、生殖腺等多個組織上皆有表現,而在輸精管上只有在末端儲精頰囊周圍的雄性腺細胞會表現IAG。在蝦苗發育期間IAG在生殖腺形成前(PL8)與性別分化期(PL12)表現量上升,且直到雄性腺生成(PL52)之前,IAG在蝦苗頭部與尾部皆有表現。接著,在摘除成熟雄蝦的雄性腺並蓄養28天後,精巢沒有發現萎縮退化的現象,而各階段生殖細胞的比例與控制組比較無顯著差異,仍有許多生殖細胞持續進行減數分裂。從其表現模式與手術的結果來推測,在白蝦上IAG可能參與生殖腺的分化或發育,但與多數甲殼類不同,在雄性發育上並非扮演關鍵性的調控因子。綜合以上,雖雄性腺摘除手術對白蝦精巢發育沒有顯著的影響,但未來仍可透過進一步的實驗探討IAG實際的功能以及自轉錄體中篩選出的潛在可能參與性別發育的基因的功能,讓我們對白蝦性別調控的機制有更多的瞭解。
Pacific white shrimp (Litopenaeus vannamei) is one of the major cultured crustaceans around the world. Female has higher growth rate and size compared with male. Therefore, monosex culture of female could increase production yield greatly. In most malacostraca crustaceans, sex differentiation is controlled by a male specific organ, androgenic gland (AG). In this study, three tissues (terminal ampule with AG attached, testis and ovary) were used to generate the cDNA library through the transcriptome analysis. 13 sex-dimorphic genes (FPKM>32 in testis/ovary, <0.213 in ovary/testis) were selected to analyze the expression profile through the qPCR analysis. One of the female-specfic gene, contig223, had exclusively expression in ovary compared to other tissues. Based on in situ hybridization data, contig223 was localized in the primary oocyte but not the oogonia. In addition, a key gene for male differentiation in most malacostraca crustacean, IAG was selected to understand the mechanism of sex differentiation in shrimp. according to qPCR data, IAG was highly expressed in the terminal ampule of vas deferens. In situ hybridization staining shows that IAG was localized in AG cell attached to the terminal ampule. In contrast, unlike Macrobrachium rosenbergii, several tissues also had IAG expression. Furthermore, IAG expression did not change in IAG-expressed tissues after the ablation of AG. Thus, our results find an ovary-specific gene (contig223) in the female shrimp. Our data also demonstrates that AG-removal male shrimp wasn’t to alter the sexual fate from male to female. Talking together, the mechanism of sex differentiation may had highly diversity between penaeid shrimp and palaemonid shrimp.
摘要 I
Abstract II
目錄 III
圖表目錄 IV
第一章、緒論 1
一、性別控制的重要性 1
1. 南美白蝦養殖產業 1
2. 單性養殖(Monosex culture) 1
二、性別的發育 2
1. 性別決定 2
2. 性別分化 3
3. 性別發育的基因調控 7
三、研究目的與方向: 9
1. 研究目的 9
2. 擬解決問題 9
第二章、性別特異性基因分析 11
一、前言: 11
二、實驗設計: 12
三、結果: 13
四、討論: 15
五、圖與表: 20
第三章、IAG基因對白蝦精巢發育的影響 30
一、前言: 30
二、實驗設計: 31
三、結果: 34
四、討論: 36
五、圖與表: 40
第四章、材料方法 54
結論 65
附錄 66
一、緒論附圖表: 66
二、補充資料: 69
參考文獻 79
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