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研究生:李琦玫
研究生(外文):Chi-Mei Lee
論文名稱:德國蜚蠊色素散佈因子之選殖及其在日週時鐘之功能解析
論文名稱(外文):Cloning and Functional Assay of pigment dispersing factor on the Circadian Clock in the German Cockroach, Blattella germanica (L.)
指導教授:李後晶李後晶引用關係
學位類別:博士
校院名稱:國立臺灣大學
系所名稱:昆蟲學研究所
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:英文
論文頁數:85
中文關鍵詞:運動日週律動神經胜肽核糖核酸干擾實驗基因靜默德國蜚蠊
外文關鍵詞:locomotioncircadian rhythmneuropeptidesRNA interferencegene silencingBlattella germanica
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在黃果蠅(Drosophila melanogaster), 馬得拉蜚蠊(Leucophaea maderae), 及黃斑黑蟋蟀 (Gryllus bimaculatus), 色素散佈因子被時鐘細胞作為輸出訊息的分子,也是使時鐘細胞能同步運行的協調因子。本論文研究是在探討色素散佈因子在德國蜚蠊的功能。首先,進行了色素散佈因子cDNA的選殖。色素散佈因子cDNA的序列及結構和小翅苯蝗 (Romalea microptera) 及寒蟬 (Meimuna opalifera) 的色素散佈因子cDNA較為相似;與黃果蠅及家蠶 (Bombyx mori) 之色素散佈因子cDNA的相似度較低。分析蜚蠊腦內色素散佈因子基因的表現型式,發現色素散佈因子基因的表現並不會呈現日週律動。此結果與黃果蠅之色素散佈因子基因的表現形式相似。色素散佈因子之前驅蛋白質是由87個胺基酸構成, 在序列及結構上仍是與小翅苯蝗及寒蟬的色素散佈因子前驅蛋白質較為相似。選殖出色素散佈因子cDNA後,就接著進行核糖核酸干擾實驗,藉由注射色素散佈因子的雙股核糖核酸來降低色素散佈因子基因的表現。從注射雙股核糖核酸的第二天起,色素散佈因子基因的表現就顯著地降低,而且這個抑制作用至少能持續56天之久。藉由組織免疫染色法,可見到實驗組蜚蠊之時鐘細胞內色素散佈因子的量明顯降低。由行為方面的研究結果可知,不論是處於有光暗週期或全暗環境的德國蜚蠊雄蟲,在注射色素散佈因子的雙股核糖核酸之後,其活動的日週律動都會消失。但同樣的處理,對原本就無法表現出活動日週律動的雌蟲,則無明顯的影響。進一步分析雄蟲的活動記錄,發現雄蟲的每日活動總量會隨著日齡的增加而增多,但注射色素散佈因子的雙股核糖核酸之後,雄蟲的活動量並未隨著其日齡的增加而明顯增多。此外,夜行性的德國蜚蠊原本在關燈後一小時內,會有一個明顯的活動高峰出現,但分析注射色素散佈因子的雙股核糖核酸後11-20天的雄蟲活動記錄,發現關燈後的活動高峰並沒有出現。若在注射後逐天檢視活動的分佈情形,可見到活動高峰在注射後日漸減低,在七天後就完全消失。由以上的結果推論,在德國蜚蠊,色素散佈因子應是時鐘細胞的訊息輸出因子,用於調控活動行為,使其表現出日週律動。關於注射色素散佈因子的雙股核糖核酸對雌蟲生殖功能的影響,也進行了初步的探討,但結果顯示並無明顯的影響。
In Drosophila melanogaster, Leucophaea maderae, and Gryllus bimaculatus, the pigment-dispersing factor (PDF) functions as output and coupling signal of locomotor circadian clocks. In this study, the German cockroach, Blattella germanica, was investigated to reveal the functions of PDF. The pdf cDNA had been cloned and its sequence and structure showed higher similarity with Romalea microptera and Meimuna opalifera than that of D. melanogaster and Bombyx mori. The pdf gene in the head of the German cockroach did not express rhythmically which displayed the similar pattern as D. melanogaster. The PDF precursor protein of the German cockroach was composed of 87 amino acids and its sequence was also similar to that of R. microptera and M. opalifera. Once the pdf cDNA had been cloned, we explored the functions of PDF in the German cockroach with RNA interference technique. After pdf double-stranded RNA (dsRNA) injection, the amount of pdf mRNA decreased significantly since the second day and this knockdown effect could persist for at last 56 days. With immunostaining technique, the clock cells of pdf dsRNA-injected cockroaches could not be stained by anti-PDF antibody. In the behavioral study, the injection of pdf dsRNA caused rhythmic males to become arrhythmic in light-dark cycles or constant darkness, but had no obvious effect on the locomotion of female cockroaches. In addition, due to the nocturnal nature of the German cockroach, the locomotor activity increased after light off or entering subjective night. However, this activity peak gradually disappeared after pdf dsRNA injection. Based on these two lines of evidences, PDF serves as an output regulator of locomotor circadian rhythm in the German cockroach. Beside, the effect of pdf dsRNA injection on the reproduction of female cockroaches had also been studied. The results showed that PDF might not be involved in the regulation of reproduction in female cockroaches.
口試委員會審定書 -i
誌謝 -ii
摘要及關鍵詞 -iii
Abstract and key words -v
Chapter 1 Introduction
1.1 The pigment dispersing factor (PDF) -2
1.2 The location of circadian clock -3
1.3 The clock genes oscillate in circadian clock -4
1.4 The pdf gene and cDNA -5
1.5 The potential functions of PDF in insects -6
1.6 The aim of this study -9
Chapter 2 Cloning and Analyzing the pdf Gene of the German Cockroach, Blattella germanica (L.)
2.1 Introduction -11
2.2 Materials and Methods -13
2.2.1 Insect -13
2.2.2 RNA extraction -13
2.2.3 Reverse transcription (RT) -13
2.2.4 3'' RACE -14
2.2.5 5'' region cloning -15
2.2.6 Genomic DNA extraction -15
2.2.7 Inverse PCR -16
2.2.8 RT-PCR for checking pdf gene expression -17
2.2.9 Data analysis -18
2.3 Results -19
2.3.1 The characterization of the pdf cDNA of the German cockroach -19
2.3.2 Structure of the pdf gene -19
2.3.3 PDF precursor protein and PDF peptide -20
2.3.4 The pdf gene expression pattern -20
2.4 Discussion -21
Tables -26
Figures -31
Chapter 3 Studying the Functions of the pdf Gene in the Circadian Rhythm of the German Cockroach, Blattella germanica (L.)
3.1 Introduction -39
3.2 Materials and Methods -42
3.2.1 Insect -42
3.2.2 RNA extraction -42
3.2.3 Reverse transcription (RT) -43
3.2.4 Construction of the template for dsRNA synthesis -43
3.2.5 RNA Interference -44
3.2.6 RT-PCR 45
3.2.7 Immunostaining 45
3.2.8 Locomotor activity monitoring 46
3.3 Results 48
3.3.1 Effects of pdf dsRNA injection on the pdf gene expression in the transcription level -48
3.3.2 Effects of pdf dsRNA injection on the PDF peptide synthesis -48
3.3.3 Effects of pdf gene silencing on circadian locomotion -49
3.3.4 Effects of pdf dsRNA injection on the reproduction of the female German cockroach - 51
3.4 Discussion -52
Tables -56
Figures -58
Chapter 4 Conclusions and General Discussion -74
References -78
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