臺灣博碩士論文加值系統

蚊子可因生殖營養週期需要血液攝食與否而分為自生型與非自生型兩種，在雌蚊的生殖營養週期，最重要的階段為卵黃蛋白合成時期，當中又以卵黃蛋白原 (vitellogenin, Vg )基因為該時期最主要且有最高表現量的關鍵基因，為了要瞭解自生型與非自生型的Vg基因表現之異同，嘗試以熱帶家蚊（Culex quinquefasciatus）與其相似卻具自生型特性之地下家蚊 (Culex pipiens molestus)作為研究材料，分析兩品種之間的Vg基因表現的模式。同時也針對蚊幼蟲生長條件的控制及生長調節藥劑 （tebufenozide）處理，探討是否能影響其成蟲生殖策略的改變，使其從非自生型轉變成自生型蚊種。結果顯示，提高幼蟲飼養的營養、降低幼蟲飼養密度，以及調整飼養光照條件，確實能增加蛹體體積，以及增大成蟲體型，但仍無法改變其生殖型式。而以 RT-PCR技術偵測蚊子的兩種Vg基因表現，結果顯示無論是地下家蚊或熱帶家蚊，其VgC1和VgC2在幼蟲及蛹期都有少量表現，但在血液攝取後及產卵前，VgC1和VgC2會有明顯的大量表現，產卵後表現量即大副降低，回復到吸血前的少量表現。然而在VgC1大量表現時期，其cDNA會有部分序列被刪除 (delete)，這個現象是否與其生殖機制調節有關，有待進一步深入研究。

Autogenous Culex pipiens molestus is a good model for better understanding the relationship between oviposition and non-blood feeding signal. Vitellogenesis is the central step which involves the synthesis and secretion of yolk protein precursors (YPP) on a massive scale by the fat body in mosquito reproduction. In this thesis, the influence of larval rearing environment and Tebufenozide treatment on the growth of mosquito were compared by using two different mosquito species, autogenous (Cx. pipiens molestus) and anautogenous (Culex quinquefasciatus). Subsequently, RT-PCR analysis of Vg mRNA extracted from Cx. quinquefasciatus and Cx. pipiens molestus mosquito during a reproductive cycle will be presented. The results showed that tebufenozide treatment, abounding larval nourishment and low larval intensity have significantly impacted on the growth character and fecundity in both mosquitoes. However, rearing environment and Tebufenozide treatment have no effect on the anautogenous of mosquito. Moreover, vitellogenin genes (VgC1, VgC2) were only highly expressed after a bloodmeal. However, VgC1 sequence showed a 61 bp deletion during the period of high expression The effect of VgC1 sequence deletion on the reproduction of these two mosquitoes is currently unknow and deserves further investigation.

致謝.......................................................................................................i
中文摘要...............................................................................................ii
英文摘要..............................................................................................iii
目錄.......................................................................................................iv
圖目錄..................................................................................................viii
表目錄....................................................................................................ix
壹、 緒言..............................................................................................1
貳、 往昔研究.......................................................................................4
一、 自生型蚊種和非自生型的蚊種..................................................4
二、 雌蚊的卵黃蛋白合成 (vitellogenesis) .......................................5
三、 胺基酸在雌蚊生殖週期中所扮演的腳色....................................6
四、 地下家蚊生殖產卵特性..............................................................7
參、 材料與方法.....................................................................................9
一、 幼蟲生長條件與成蟲生殖性能之關係........................................9
（一） 蚊蟲來源與飼養.....................................................................9
（二） 不同飼料種類餵食對於幼蟲生長的影響..............................9
（三） 不同飼料量餵食對於幼蟲生長的影響.................................10

（四） 地下家蚊幼蟲養份攝取情形與成蟲吸血行為之關連..........10
（五） 生長空間密度對於幼蟲生長之影響....................................11
（六） Tebufenozide處理幼蟲對於成蟲之影響..................................11
（七） 光照強度對成蟲的壽命影響...................... ...................................12
二、 熱帶家蚊與地下家蚊卵黃蛋白基因的表現...............................12
(一) 引子的測試..........................................................................13
(二) 卵黃蛋白基因於地下家蚊及熱帶家蚊各生長期之表現.......13
(三) Tebufenozide對於Vg基因表現的影響................................13
1. RNA的萃取.......................................................................14
2. cDNA的合成.....................................................................14
2.1 primer的設計.................................................................14
2.2反轉錄酶反應 (Reverse Transcriptase, RT) ....................15
2.3聚合酶鏈鎖反應 (Polymerase chain reaction, PCR).........16
3. PCR產物的分析.................................................................16
4. PCR產物定序.....................................................................16
4.1 DNA膠體純化萃取..........................................................16
4.2 DNA接合反應(Ligation) .................................................17
4.3 質體轉型(Transformation) ..............................................17
三、 以二維電泳技術分析地下家蚊行自生型生殖週期時，產卵前後之腹部蛋白變化..................................................................................17
(一) 雌蚊腹部取樣與蛋白質製備.................................................18
(二) 二維電泳...............................................................................18
肆、 結果.................................................................................................19
一、 幼蟲生長條件與成蟲生殖性能之關連.........................................19
(一) 不同飼料種類餵飼對於幼蟲生長的影響.....................................19
(二) 不同飼料量餵食對於幼蟲生長的影響....................................19
(三) 地下家蚊幼蟲養份攝取情形與成蟲吸血行為之關連....................20
　　 (四) 生長空間密度對於幼蟲生長的影響.......................................21
（五）Tebufenozide處理幼蟲對於成蟲之影響................................22
(六) 光照強度對成蟲的影響............................................................23
二、 熱帶家蚊與地下家蚊卵黃蛋白基因的表現.................................23
(一) 引子對的測試..........................................................................23
(二) 熱帶家蚊的Vg基因表現模式.................................................25
(三) 地下家蚊的Vg基因表現模式.................................................25
(四) Tebufenozide對於熱帶家蚊Vg基因表現的影響....................26
三、 以二維電泳技術分析地下家蚊行自生型生殖週期時，產卵前後之 腹部蛋白變化............................................................................27
伍、 討論................................................................................................29
陸、 參考文獻........................................................................................34
柒、 附錄...............................................................................................37
附錄一、血液攝食驅動卵黃生成示意圖..............................................37
附錄二、以Ace-orthologous acetylcholinesterase (Ace) 基因進行熱帶家
蚊及地下家蚊之鑑定............................................................38

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