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研究生:左雨涵
研究生(外文):Yu-Han Zuo
論文名稱:東方果實蠅成蟲脂肪體之特性與芳基蛋白受器基因及卵黃蛋白基因1之轉錄調節
論文名稱(外文):Characteristics of fat body and transcriptional regulation of arylphorin receptor gene and yolk protein gene 1 in the adult oriental fruit fly, Bactrocera dorsalis
指導教授:路光暉
指導教授(外文):Kuang-Hui Lu
學位類別:碩士
校院名稱:國立中興大學
系所名稱:昆蟲學系所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:64
中文關鍵詞:脂肪體芳基蛋白受器cDNA篩減法激素
外文關鍵詞:fat bodyarylphorin receptorhormone
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脂肪體於昆蟲體內負責儲存營養、提供能量及代謝物質。於實體解剖與組織切片結果顯示,東方果實蠅(Bactrocera dorsalis (Hendel))成蟲腹部具有兩種形式之脂肪體,一為白色顆粒球狀的幼蟲形式脂肪體(larval fat body),另一為扁平片狀的成蟲形式脂肪體(adult fat body)。隨著成蟲的成長,幼蟲形式脂肪體所佔比例逐漸遞減,而成蟲形式脂肪體則逐日增加。利用cDNA篩減法(cDNA subtraction)篩減第0日齡及第9日齡成蟲腹部脂肪體各自專一表現之基因,其結果顯示第0日齡成蟲腹部脂肪體表現之基因中,以芳基蛋白受器基因(arylphorin receptor gene, ArR)所佔比例最高,達15%;而第9日齡者,則以卵黃蛋白基因1(yolk protein gene 1, yp1)最多。以RT-PCR分析,芳基蛋白受器基因大量表現於幼蟲形式脂肪體;相對地,卵黃蛋白基因1於幼蟲形式脂肪體不表現,僅表現於雌成蟲的成蟲形式脂肪體。
正常情況下,隨著東方果實蠅成蟲成長,芳基蛋白受器基因表現量逐漸降低,而卵黃蛋白基因1表現量則為增加。以飢餓處理,芳基蛋白受器基因及卵黃蛋白基因1的表現量較正常飼育少。以青春激素類似物-百利普芬(pyriproxyfen)處理,芳基蛋白受器基因的表現量略少於對照組;但卵黃蛋白基因1於百利普芬處理後的第16小時即開始表現。以蛻皮激素(20-hydroxyecdysone, 20E)處理,可延長芳基蛋白受器基因的表現時間;但於卵黃蛋白基因1表現量則無顯著影響。以早熟素(precocene II)抑制青春激素,芳基蛋白受器基因表現量較對照組高;卵黃蛋白基因1表現量則較對照組略低。若先以早熟素處理,再以百利普芬處理後,芳基蛋白受器的基因表現量較對照組略高;但卵黃蛋白基因1表現量與對照組無明顯差異。
蛻皮激素及早熟素可刺激芳基蛋白受器基因表現,但百利普芬卻會抑制芳基蛋白受器基因的表現;蛻皮激素不影響卵黃蛋白基因1表現,而百利普芬可刺激卵黃蛋白基因1的表現,但早熟素會降低芳基蛋白受器基因的表現量。
Insect fat body is responsible for storing nutrients, providing energy and metabolizing materials. Dissection and histological observation revealed that there were two forms of fat body in the adult abdomen of Bactrocera dorsalis (Hendel), i.e., white nodule-like “larval fat body” and thin sheet-like “adult fat body”. The amount of larval fat body was decreasing while adult fat body was increasing following adult growth. We further attempted to screen stage specific-expressed genes from the abdominal fat body of 0-day-old and 9-day-old adults using cDNA subtraction. The subtracted results showed that arylphorin receptor (ArR) gene expressed most abundantly in the 0-day-old adult fat body, about 15%; yet yolk protein gene 1 (yp1) was the major gene expressed in 9-day-old adult fat body. RT-PCR analysis demonstrated that ArR expressed greatly in larval fat body; in contrast, yp1 did not express in larval fat body, but specifically expressed in adult fat body of adult females.
The transcriptional expression of ArR was decreasing, but yp1 was increasing with normal adult growth. In adult fat body, ArR and yp1 expressed less in starved females than that of control females. The duration of transcriptional expression of ArR was extended but that of yp1 was similar to the control after 20-hydroxyecdysone (20E) treatment. The expression of ArR was little lower after juvenile hormone analog, pyriproxyfen, treatment than control, but yp1 expressed earlier in the adult females at 16 h after pyriproxyfen treatment. ArR expressed higher, but yp1 expressed lower after an anit-juvenile hormone agent, precocene II, treatment than control. ArR expressed higher after precocene II and pyriproxyfen treatment than control, but the expression of yp1 after was similar to control.
20E and precocene II induced ArR expression that was inhibited by pyriproxyfen. There was no obvious effect of 20E on the expression of yp1 which could be induced by pyriproxyfen, but precocene II inhibited the expression of yp1.
一、中文摘要------------------------------------------------------------------------ 1
二、英文摘要------------------------------------------------------------------------ 3
三、前言------------------------------------------------------------------------------ 5
四、前人研究------------------------------------------------------------------------ 7
(一) 脂肪體之分布及功能---------------------------------------------------- 7
(二) 成蟲時期脂肪體之生成------------------------------------------------- 9
(三) 饑餓及激素對脂肪體消長之影響------------------------------------ 10
(四) Hexamerin與芳基蛋白受器-------------------------------------------- 11
(五) 芳基蛋白受器之功能--------------------------------------------------- 12
五、材料與方法------------------------------------------------------------------- 15
(一) 試驗昆蟲之飼育-------------------------------------------------------- 15
(二) 冷凍切片----------------------------------------------------------------- 15
(三) 收集脂肪體樣本-------------------------------------------------------- 15
(四) 製備總量RNA----------------------------------------------------------- 16
(五) 純化mRNA-------------------------------------------------------------- 17
(六) cDNA篩減法------------------------------------------------------------- 18
(七) DNA電泳----------------------------------------------------------------- 21
(八) 聚合酶連鎖反應產物之選殖----------------------------------------- 21
(九) 飢餓及激素處理與基因表現量測定-------------------------------- 23
六、結果---------------------------------------------------------------------------- 25
(一) 東方果實蠅成蟲腹部脂肪體之形態與切片之觀察-------------- 25
(二) cDNA篩減法之結果---------------------------------------------------- 26
(三) 芳基蛋白受器基因之分析-------------------------------------------- 27
(四) 芳基蛋白受器基因表現模式與飢餓或激素對其表現之影響-- 27
七、討論---------------------------------------------------------------------------- 30
(一) 東方果實蠅成蟲腹部幼蟲與成蟲形式脂肪體之形態、消長與功能--------------------------------------------------------------------------- 30
(二) 芳基蛋白受器基因----------------------------------------------------- 32
(三) 芳基蛋白受器與脂肪體消長之關係-------------------------------- 32
(四) 激素對芳基蛋白受器基因表現之影響----------------------------- 33
(五) 結語----------------------------------------------------------------------- 34
八、參考文獻---------------------------------------------------------------------- 35
九、圖表---------------------------------------------------------------------------- 41
十、附錄---------------------------------------------------------------------------- 61
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