阿拉伯芥生物時鐘的功能與可塑性研究

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林岑穎(TSEN-YING LIN) 查詢紙本館藏

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論文名稱

阿拉伯芥生物時鐘的功能與可塑性研究
(The function and tunability of circadian clock in Arabidopsis)

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至系統瀏覽論文 (2024-7-19以後開放)

摘要(中)

阿拉伯芥的生物時鐘由許多基因組成的多條回饋迴路所構成，以每24小時為一循環運作。環境會調控大部分生物時鐘基因的表現，也因此使植物可以藉由日夜光線變化推斷時辰以適應環境。生物時鐘亦具有強力的自我維持性讓其可持續以每24小時為一日的節律運轉而不受環境波動或缺少日夜交替的影響。我對當外界日長與內在生物時鐘節律不相合時，植物生物時鍾是否具有配合外界調整其節律與推測時間的可塑性感興趣，我將Col-0與lwd1lwd2生長於15小時白天15小時夜晚(15L/15D)，12小時白天12小時夜晚(12L/12D)或6小時白天6小時夜晚(6L/6D)下觀測生長情況。結果顯示，在6L/6D下，由於CONSTANS(CO)表現隨日長改變，使FLOWERING LOCUS T(FT) 累積在Col-0增加，但於lwd1lwd2反而減少，因此，相較於生長在12L/12D環境下，在6L/6D下生長的Col-0會提早開花，但是lwd1lwd2會晚開花。我的研究也發現：生物時鐘基因的表現週期無論在Col-0或lwd1lwd2中均可變動以配合外界日長，但每個基因表現相位可塑性並不大。而當外界缺乏日夜輪替時，內在生物時鐘會在一天內回復其內生週期。我們推測植物生物時鐘具有的很強的穩健性，同時生物時鐘基因表現亦受晝夜循環調控，其週期可以配合日長，使植物得以迅速適應環境又不致於過於受到環境擾動的影響。
另外，LIGHT REGULATED WD1/2 (LWD1/2) 為調節生物時鐘的兩個重要時鐘基因。雖然先前對LWD1在生物時鐘的角色已有較深入的研究，但目前對LWD2的了解仍停留在其與LWD1功能冗餘的推測。我利用LWD2-Flag表現於lwd1lwd2突變株中發現LWD2可回復突變株早花的表現型與生物時鐘基因提早表現到峰值的情況，所以認定LWD2功能與LWD1相似。

摘要(英)

In Arabidopsis thaliana, genes in several interlocked feedback loops constitute the circadian clock and function to generate a 24-h rhythmic oscillation, enabling plants to anticipate and adjust themselves to daily environment. The expression of most clock components is regulated by light, making the system highly sensitive to changes in light conditions. On the other hand, circadian clock is also very robust, allowing plants to maintain the rhythm for counting the time correctly when exogenous time cues are disturbed or deprived. I was interested in examining whether plants possess plasticity to adjust their circadian clock when external time cues do not match the internal one. I monitored how the environmental light regimes could impact the plant growth and development by growing Col-0 and lwd1lwd2 directly under 15-h light (L)/15-h dark (D), 12L/12D or 6L/6D conditions. Results showed Col-0 flowered early while lwd1lwd2 delayed the flowering time, contributed by the expression patterns of CONSTANS (CO) modulated under various L/D cycles, therefore leading to the slightly increased expression of FLOWERING LOCUS T (FT) in Col-0 but significantly decreased in lwd1lwd2. Circadian oscillator in both Col-0 and lwd1lwd2 were tunable to certain degree when facing various day lengths. When plants were released into free-running conditions, the internal clock would restore its dominating role in regulating clock gene expression. This suggested that plants are versatile to fit circadian period to various light/dark length, yet maintain a very robust internal circadian clock. The second part of my thesis focused on the study of a clock protein LWD2. LWD1/2 were previously characterized to be key regulators of the central circadian clock. Though the functions of LWD1 were previously characterized, the current understanding of LWD2 remains to be speculated to be functional redundant to LWD1. My study found that LWD2-Flag could fully complement the mutant phenotype of lwd1lwd2, suggesting LWD2 has similar function as a LWD1.

關鍵字(中)

★ 阿拉伯芥
★ 生物時鐘

關鍵字(英)

★ arabidopsis
★ circadian clock

論文目次

中文摘要 i
ABSTRACT ii
Table of Contents iv
Table of Tables vi
List of Figures vii
List of Appendices viii
Introduction 1
Materials and Methods 5
I.Plant Materials and growth conditions---------------------------------------5
II.Flowering time measurement -----------------------------------------------6
III.RNA Isolation --------------------------------------------------------------6
IV.Reverse Transcription PCR -------------------------------------------------9
V.qPCR ---------------------------------------------------------------------10
VI.Bioluminescence measurement and analysis ------------------------------12
VII.LWD2 peptide antibody generation ---------------------------------------13
VIII.Protein extraction and western blot analysis -----------------------------13
Results and discussion ---------------------------------------------------18
1.Col-0 early flowered under 6L/6D conditions---------------------------18
2. The expression of central oscillator genes is under the control of both circadian clock and light/dark signals ---------------------------------------19
3. Oscillator genes show different degrees of tunability under non-24-h day length in Col-0 -------------------------------------------------------------21
4. The endogenous circadian rhythm is highly robust -----------------------22
5. llwd11llwdd22 can compensate the early flowering phenotype under 6L/6D--------------24
6. The oscillator can anticipate external time correctly when day length fits the 18-h circadian rhythm circadian period length of lwd1lwd2 ---------------------------------24
7. The expression of central oscillator genes in lwd1lwd2 also showed tunability under various day length-----------------------------------------------------------------------------25
8. The oscillator cannot precisely anticipate external time when the day length and endogenous rhythm were decoupled in llwd1lwd2 ---------------------------------------26
9. lwd1lwd2 still possessed a robust 18-h endogenous rhythm ----------------------------27 10. LWD2 alone could rescue the early flowering phenotype and clock defect in
lwd1llwdd22 ---------------------------------------------------------------------------------------28 11. Antiserum against LWD2 recognized both LWD2 and LWD1 ------------------------29
Conclusion 29
References 31
Tables 33
Figures 34
Appendices 50

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指導教授

吳素幸(SHU-HSING WU)

審核日期

2019-7-23

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