李後晶臺灣大學：昆蟲學研究所吳佳恩Wu, Chia-EngChia-EngWu2007-11-262018-06-292007-11-262018-06-292005http://ntur.lib.ntu.edu.tw//handle/246246/55033日週律動現象普遍存在於生物當中，從原生動物到哺乳動物皆有一個近24小時的律動週期。果蠅的相關研究指出，生物時鐘的分子機制是由一個負回饋的機制所調控，時鐘細胞中period (per) 以及timeless (tim) 基因所產生的PER及TIM蛋白會抑制自身基因的表現。如同果蠅，其他昆蟲物種包括德國蜚蠊 (Blattella germanica)，per基因的表現具有日週律動的現象。在活動行為方面，德國蜚蠊的雄成蟲具有日週律動，但德國蜚蠊雌成蟲的活動行為受到生殖週期的影響，不具有日週律動的表現，此現象稱為掩蓋效應 (masking effect)。我們推測，生殖系統中的周邊時鐘 (peripheral clock)，會影響位於腦中的主要時鐘 (master clock) 對活動行為日週律動的表現。雙紋姬蠊 (B. bisignata) 是德國蜚蠊的近緣種，其雌蟲的活動行為日週律動表現不受生殖週期的影響，不具有掩蓋效應。本篇研究雙紋姬蠊與德國蜚蠊的時鐘基因per在頭部以及生殖系統中睪丸、卵巢與副腺的表現情況，試圖解釋掩蓋效應與日週律動的關係。選殖出的雙紋姬蠊per cDNA序列與德國蜚蠊per cDNA序列具有高度的相似性。根據Real-Time RT PCR的結果，per mRNA在雙紋姬蠊與德國蜚蠊的雄蟲頭部與部分生殖組織當中皆有相似的表現，其表現量在夜晚達到高峰。此外，per mRNA在雙紋姬蠊與德國蜚蠊雌蟲頭部與卵巢的表現顯示，per基因在這些組織當中似乎扮演另一種的調控角色，而與雄蟲頭部的表現情況不同。Circadian rhythms are nearly 24-hour rhythms of biological processes that persist under constant environmental conditions. These rhythms have been described in a variety of organisms ranging from prokaryotes to mammals. In Drosophila, the driving force underlying the circadian system is the pacemaker cells with time signal generated by the interactions of the negative feedback loop of period and timeless genes. In several other insect species, including the German cockroach, per expression undergoes circadian fluctuations. Oddly, the overt rhythm of locomotion of female German cockroach is masked by its own reproductive cycle. We suspect that clocks in peripheral tissues, especially in reproductive organs, may affect the brain-clock (master clock) which has been demonstrated to control the locomotor circadian rhythm. However, the Blattella bisignata, a closely-related species of the B. germanica, expresses locomotor circadian rhythm without masking effect in both sexes. This study focuses on the expression of per in brain and reproductive tissues in both species to unveil the possible interaction of clock gene expression under masking effect of locomotor circadian rhythm. Period gene in the double-striped cockroach has been cloned and its partial sequence shows a high degree of homology with the one from the German cockroach. Furthermore, the expression patterns of period mRNA were similar in heads and reproductive tissues, which were peaked at night. The exceptions in expression patterns of testes of German cockroach and ovaries of both species suggest that different regulatory clock mechanisms maybe involved.Table of Contents Literature review……………………………………………………………..1 Molecular clock……………………………………………………………1 Master and peripheral clocks…………………………………………………..5 Comparison between Blattella bisignata and B. germanica……………...10 Calling and mating behavior of the female German cockroach………...11 Reproductive system of the male Blattella germanica………………….12 Posttranslational regulation of PER……………………………………...14 Introduction…………………………………………………………………...17 Materials and Methods……………………………………………………….20 Insects………………………………………………………………………20 Reverse transcription (RT)-PCR………………………………………….20 Subcloning and sequencing………………………………………………..21 Rapid amplification of cDNA ends (RACE)……………………………...22 Computer analysis………………………………………………………….22 Real Time RT-PCR…………………………………………………………22 Western blotting…………………………………………………………….23 Results………………………………………………………………………….26 Cloning of period cDNA from Blattella bisgnata………………………..26 Sequence homology and structure analysis of PER………………………27 Temporal expression of period mRNA in heads and reproductive tissues of Blattella germanica…………………………………………………………29 Temporal expression of period mRNA in heads and reproductive tissues of Blattella bisignata…………………………………………………………..30 Temporal expression of PER in heads and reproductive tissues of Blattella germanica……………………………………………………………………32 Discussion………………………………………………………………………33 References………………………………………………………………………41 Appendix………………………………………………………………………..74 Acknowledgements…………………………………………………………….86647604 bytesapplication/pdfen-US時鐘基因clock gene periodthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/55033/1/ntu-94-R91632003-1.pdf