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Title: | 德國蜚蠊之時鐘基因機制解構與探討 Unveiling the circadian clockwork of the German cockroach Blattella germanica |
Authors: | Chi-Yuan Huang 黃啟原 |
Advisor: | 李後晶(How-Jing Lee) |
Keyword: | 日週律動,時鐘基因,德國蜚蠊, circadian clock,period,cryptochrome,timeless, |
Publication Year : | 2017 |
Degree: | 碩士 |
Abstract: | 日週節律是由內生性時鐘基因所形成之負轉錄回饋機制構成的日週時鐘所調控。從細菌、昆蟲到人,幾乎所有生物中皆具有日週節律的現象並且表在生理、代謝與行為中表現。至今已有大量關於果蠅(Drosophila melanogaster)時鐘機制的研究,然而在非模式昆蟲物種中,日週時鐘的分子機制的組成可能有所不同,例如帝王蝶(Danaus plexippus)在研究中已顯示與果蠅(D.melanogaster)不完全相同。在前人研究中,德國蟑螂NGS數據庫中發現了類果蠅隱色素(Bgcry1)和類哺乳動物隱色素(Bgcry2)。利用基因干擾技術(RNA interference)減低Bgcry2的表現顯示蟑螂的活動及cycle(Bgcyc)基因的表現量皆沒有日週律動,然而period(Bgper)和cycle(Bgcyc)基因的表現量上升。在本研究中利用HEK293T細胞株,進行共免疫沉澱(co-Immunoprecipitation),GST下拉測定(GST-Pulldown Assay)和鄰近連接測定(Proximity Ligation Assay)來研究時鐘基因的蛋白質間之交互作用與作用於細胞的位點。首先我們將Bgcry2,BgPer與老鼠(Mus. musculus)基因比對發現其具有抑制負轉錄回饋機制之功能。在本實驗我們提出蟑螂兩種的轉錄回饋機制,其一為,BgCRY2與細胞質中的BgTIM結合,並由BgTIM將其帶入細胞核中。第二條路徑為BgTIM與細胞質中的BgPER相互作用,然而BgPER與BgTIM分別進入細胞核。雖然德國蟑螂的日週時鐘機制建構在果蠅與帝王蝶模型之上,本篇研究主要不同為BgTIM與BgCRY2的結合並涉及時鐘基因之負轉錄回饋機制,且BgPER、BgTIM為分開進入細胞核中。這個新的假設仍然需要進一步的調查來驗證晝夜節律鐘的完整性。 A circadian rhythm is controlled by an endogenous circadian clock which is driven by autoregulatory transcription feedback loop of clock genes. This behavioral or physiological phenomenon repeats itself approximately 24 hours and commonly expresses in almost all organisms from bacteria to human. The molecular mechanisms of insect circadian clock have been well studied in fruit fly (Drosophila melanogaster). However, the components of clockwork may be different in non-model insects since some differences have been reported in monarch butterfly (Danaus plexippus). In previous studies, both Drosophila-like cryptochrome (Bgcry1) and mammalian-like cryptochrome (Bgcry2) were found in the German cockroach form the next-generation sequencing data. By knocking down Bgcry2 gene with RNA interference shows arrhythmicity in locomotion and expression level of cycle (Bgcyc) (a transcription factor of the circadian feedback loop), but mRNA levels of period (Bgper) and cycle (Bgcyc) genes are increased. In this study, I investigated the protein-protein interaction of circadian clock by performing co-immunoprecipitation, GST-pulldown assay and proximity ligation assays. These results show that BgCRY2 binds with either BgTIM or BgPER in the cytoplasm of HEK293T cells, while BgTIM interacts with BgPER in the cytoplasm. In combination of previous studies and present results, I propose a model in which BgTIM and BgPER form a complex and accumulate in the cytoplasm, and then disassociates before entering the nucleus. But BgPER may be independently translocated into the nucleus to act as a repressor of clock genes. Another mode is also proposed that BgCRY2 is carried by BgTIM to enter the nucleus and BgCRY2 may be the repressor based on the alignment results. I also suggest that a photoreceptor BgCRY1 may be a photoreceptor, expressing in the cytoplasm, to bind to BgTIM for degrading BgTIM during photophase. Although the clockwork of the German cockroach is largely following the Drosophila model, the minor difference is BgTIM, BgCRY2 and BgPER involved in an autoregulatory feedback loop and at least two modes of feedback loop involved in the circadian clockwork of the German cockroach. The BgTIM/BgCRY2 may enter nucleus for repression of their own transcription is a new finding in this study. This new hypothesis requires further investigation to verify the completeness of the circadian clockwork. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/68391 |
DOI: | 10.6342/NTU201703863 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 昆蟲學系 |
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