- 學位論文
透過阿拉伯芥自然品系之全基因組關聯分析探討生物時鐘節律高溫補償之調控
Genome-wide association study of temperature compensation of circadian rhythms in Arabidopsis thaliana natural accessions
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摘要
生物時鐘為生物體重要的生理調控機制，可驅使生理活動在合適的時間點運行。由於地球自轉週期為24小時，因此生物的生物時鐘週期也大約為24小時。生物時鐘具有一個稱為溫度補償的特性，當環境溫度變化時，能讓生物時鐘週期的長度維持在一個相對穩定的範圍內，以保持生物時鐘計時功能的穩定性。近年來，雖已有前人研究找到可在高溫下穩定生物時鐘週期長度的基因，然而此方面的研究仍然有限。因此，我希望透過全基因組關聯分析（GWAS）來找尋新的調控者。GWAS透過運算性狀與不同基因型間之單一核苷酸多型性（SNP）的關聯性強度，來篩選可能參與調控性狀之基因。為了收集性狀資料，我建立了葉片開合影像偵測系統以收集生物時鐘週期資料，並挑選四十個具有豐富基因多樣性的伊比利半島阿拉伯芥自然品系作為植物材料。我分別檢測了這四十個自然品系在22度下及29度下的生物時鐘週期長度，發現無論是在哪個溫度下，品系之間的週期長度存在明顯的差異。此外，不同品系也展現出了不同的溫度補償能力，實驗結果顯示有20個品系對於高溫比較敏感，當溫度變化時，週期長度會有顯著性的不同，顯示這些品系將週期補償回來的能力較弱；而另外20個品系具備較強的溫度補償能力，在轉移到高溫下後，週期長度較能維持在一個相對穩定的範圍內。實驗結果也顯示，當被轉移到高溫下時，不同品系的週期變化方向也會有所不同，在這20個對溫度較敏感的品系中，有15個品系的週期會變短，而有5個品系的週期會有加長的現象。雖然透過GWAS的分析，並沒有找到與高溫下週期變化有顯著關聯性的SNP，然而我有搜索到數個GWAS分數較高、會造成非同義突變的SNP，在未來，可以進一步去偵測這些SNP所坐落之基因的突變株性狀，以篩選出可能的調控者。另外，我也觀察到品系的週期資料與數個地理環境因子具有顯著的相關性，顯示生物時鐘系統中的功能差異，可能會對於品系去適應其當地環境有所助益。總結以上，GWAS的分析揭示了數個具有潛力的基因，可待未來進一步檢測其生物時鐘性狀，而為適應不同的地理環境，伊比利半島自然品系可能各自演化出了適合其生存的生物時鐘功能。
並列摘要
Circadian clocks act as crucial regulators to switch on or off biological events in proper timing in plants. The clock rhythm is entrained by daily environmental changes to roughly 24 hours. A crucial property of circadian clocks, called temperature compensation, can keep the clock periods relatively steady over temperature change to accommodate environmental temperature changes. In recent years, several temperature-compensation factors for maintaining clock rhythms at high temperature have been identified. However, factors for regulating temperature compensation still remain to be explored. Here, I used genome-wide association study (GWAS) to analyze the correlations between period changes and single-nucleotide polymorphisms (SNPs), trying to reveal potential regulators. To obtain period data, I set up a leaf movement imaging system which can detect clock rhythms by analyzing the open-and-fold configuration of leaves. With high genetic diversity, 40 Arabidopsis thaliana Iberian natural accessions were selected, and the clock periods of the accessions were measured at both 22℃ (normal temperature) and 29℃ (high temperature). The period lengths of the 40 accessions varied at the same temperature condition, 22℃ or 29℃, implying the diverse clock regulation among the accessions. Furthermore, the sensitivities to temperature changes were also different within the accessions. The results showed that 20 accessions were relatively sensitive to high temperature. Their period lengths were significantly different between 22℃ and 29℃, while the period lengths remained relatively constant in the other 20 accessions. Moreover, the directions of period shifting were also different. Among the 20 relatively sensitive accessions, the period lengths of 15 accessions shortened at high temperature, while 5 accessions exhibited longer period lengths after being transferred to high temperature. Despite the diverse period data among the accessions, no SNP was significantly associated with the period changes through GWAS analyses. Even though no significant association was identified, several non-synonymous SNPs with relatively high GWAS scores were found, and further measurements of clock traits could still be carried out on those SNP-located genes for preliminary validations. I also found that the diverse period lengths at both 22℃ and 29℃ correlated with several environmental variables, indicating that the diverse clock regulation might contribute to the adaptation of the Iberian accessions. To sum up, GWAS analyses revealed several potential components which may be worth further investigation, and the diverse clock regulation might adapt the Iberian accessions to their local environment.
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