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研究生:林冠華
研究生(外文):George Guan-Hua Lin
論文名稱:德國蜚蠊生物時鐘週期基因之選殖與其表現分析
論文名稱(外文):Cloning of a circadian clock gene period from the German cockroach, Blattella germanica (L.) and the analysis of its expression
指導教授:李後晶李後晶引用關係劉瑞芬劉瑞芬引用關係
指導教授(外文):How-Jing LeeRuey-Fen Liou
學位類別:碩士
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:2000
畢業學年度:88
語文別:英文
論文頁數:28
中文關鍵詞:生物時鐘日週律動週期基因德國蜚蠊
外文關鍵詞:biological clockcircadian rhythmperiodBlattella germanica
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生物時鐘廣泛存在於生物中。關於生物時鐘的分子機制之研究,近幾年來進展的非常迅速。從目前最重要的兩個研究物種:黃果蠅與老鼠。我們知道時間訊息的中樞是由層層相扣的負回饋機制所組成。雖然這樣的機制在黃果蠅或是老鼠都存在,而且參與其中的分子亦彼此具有演化上的保守性,但是這些分子的表現狀況與調控方式卻不盡相同。分子是如何地演化而造成這樣的差異,始終是生物時鐘研究者的疑惑。解答這樣的問題,將會使我們對於時鐘分子機制的基本性質有更深一層的了解。要回答這樣的問題,擴展研究的物種對象,透過比較的方式,是唯一的途徑。我們利用 Touch-down RT-PCR 與 RACE 的方式選殖出德國蜚蠊生物時鐘基因-週期 (period)。分析此段核酸序列所推演出的胺基酸序列,我們發現德國蜚蠊 PER 蛋白質展現出一些特有的序列保守性,而這些序列的保守性是先前不曾被發現的。PER 蛋白質序列最早由 Colot 等人根據數種果蠅的PER 蛋白質序列定義出六個重要保守區。目前的研究幾乎認為,所有昆蟲的PER 蛋白質只在第一到第四區具有共通的保守性,而第五與第六區的保守性則為雙翅目昆蟲所特有。但是令人感到訝異的是,德國蜚蠊的PER 蛋白質除了可以找到第一到第四區的保守性,在第五與第六區也與雙翅目的PER 蛋白質共同具有保守性。這樣的的結果不禁讓我們懷疑第五與第六保守區是否是廣泛存在於昆蟲界。此外,對於第五與第六保守區在PER 蛋白質中所扮演的功能亦值得重視。除此之外,德國蜚蠊的PER 蛋白質在第六區之後仍具有126個胺基酸。這些胺基酸序列並未在目前已知的昆蟲PER 蛋白質中發現,而令人更驚喜的是,此段序列與老鼠的PER 蛋白質具有相似性,這是之前未曾發現的。關於這一相似區的功能,目前並不是很清楚,但卻值得相關研究人員重視。相信這些新發現的序列保守性,將會促使我們對於生物時鐘的分子機制有更深入的了解。除此之外,我們亦藉由 RNase Protection Assay 方法測量德國蜚蠊per mRNA 的表現,證實其表現具有生物時鐘特性。表現分析的結果更加地支持我們真的是選殖出德國蜚蠊生物時鐘基因-週期 (period)。
Interlocked feedback loop manifests the endogenous circadian clock that synchronizes itself to the daily cycles and controls the overt rhythm in biochemical, physiological, and behavioral performances. Studies of the circadian molecular mechanisms in fruit fly and mouse reveal that homologous molecules shared and used by different organisms, but their expression and regulation are not similar. To bridge the gap and find out the missing link in the molecular evolutionary process, delving into unexplored species is essential. The German cockroach Blattella germanica was well documented in the study of circadian rhythmicity; of special interest, the circadian locomotor expression in female adult was masked by the development of ovaries. However the characteristics of the masking mechanism are not clear yet. As the first step to delve into the molecular masking mechanism and to explore the differences of PER proteins from diverse organisms, cDNA of the circadian clock gene period was cloned from B. germanica by touch-down RT-PCR and RACE. Sequence analysis indicates it encodes an open reading frame of 1214 amino acids. Interestingly, the PER protein of B. germanica (bPER) displays significant homology to the PER protein of Drosophila melanogaster (dPER) in the c1 to c6 regions, which were defined by Colot et al., (1988); however, PER proteins from other organisms except Dipteran insects don''t exhibit any homology to dPER in the c5 and c6 regions. In addition, the amino acid residues downstream of the c6 region of bPER exhibit significant homology to mouse PER proteins (mPER) in the C-terminus, and this homology is unique to other PER proteins from Insecta. Although some characteristics of PER proteins performing in the clockwork have been known, these new homologies found in bPER to dPER and mPER might lead us to consider if PER proteins display other unknown functions in the clockworks, especially other unknown output signal pathway. Temporal expression analysis by the RNase Protection Assay (RPA) method in male adults indicates that the expression of per mRNA from B. germanica displays circadian fluctuation. This additionally proves that the per cDNA obtained in this study is the circadian clock gene period from B. germanica.
Table of Contents
Introduction………………………………………………………………………. 1
Materials and Methods…………………………………………………………... 5
Insects…………………………………………………………………………. 5
Touch-down RT-PCR………………………………………………………… 5
Subcloning and sequencing…………………………………………………... 5
Rapid amplification of cDNA ends………………………………………… .. 6
RNase proteciton assay………………………………………………………. 6
Northern blotting……………………………………………………………... 7
Computer analysis…………………………………………………………… 7
Results……………………………………………………………………………... 8
Cloning of per cDNA from Blattella germanica…………………………….. 8
Sequence homology of bPER………………………………………………… 8
Protein structure analysis of bPER………………………………………… .. 17
per mRNA oscillates in male German cockroach………………………… .. 18
Discussion………………………………………………………………………... 21
References………………………………………………………………………… 25
Acknowledgements……………………………………………………………….. 28
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