DSpace collection: 博碩士論文

台灣藍綠菌Cyanobacterium aponinum在高光逆境下自發產生的耐高光突變株全基因組定序與表型分析;Whole-genome sequencing and phenotypic analysis of spontaneously generated high-light-resistant mutant strains of the thermotolerant Taiwanese cyanobacterium Cyanobacterium aponinum

Fri, 06 Mar 2026 10:24:39 GMT

title: 台灣藍綠菌Cyanobacterium aponinum在高光逆境下自發產生的耐高光突變株全基因組定序與表型分析;Whole-genome sequencing and phenotypic analysis of spontaneously generated high-light-resistant mutant strains of the thermotolerant Taiwanese cyanobacterium Cyanobacterium aponinum abstract: 本研究探討藍綠菌Cyanobacterium aponinum在極端高光逆境下的耐受表型演化與光保護策略。結果顯示，野生型菌株在1200 μmol photons m⁻² s⁻¹ 的高光條件下大部分生長停滯並出現色素降解，僅少數細胞能存活並恢復增殖，突顯其在光系統與調控網路中存在快速適應的潛力。通過篩選與培養耐高光突變株，發現其光系統運作、能量耗散與抗氧化能力均重新達到平衡，且在持續光條件下生長速率與野生型相當，顯示高光適應並未伴隨顯著生長成本。全基因組分析顯示，耐高光突變株僅累積少量突變事件，其中C. aponinum AL20115HLr-2的rpaA基因發生9 bp插入，導致三個胺基酸插入，可能是驅動高光耐受的核心事件。RpaA作為晝夜節律輸出型轉錄因子，可調控光合作用、碳代謝與修復系統，其插入突變可能透過微調二聚化穩定性及DNA結合親和力，改變下游數百個基因的轉錄輸出，實現對光逆境的快速適應。色素分析顯示，突變株類胡蘿蔔素顯著上升，葉綠素a含量下降，呈現典型耐高光光合生物的色素重編程模式，透過「降低能量輸入 + 提高光保護機制」的策略，降低光氧化傷害。儘管突變株在光暗循環下生長速率下降，顯示晝夜節律輸出可能受 rpaA 插入突變影響，但其在高光環境下的存活優勢仍明顯。綜合而言，C. aponinum AL20115HLr-2 的耐高光表型可能是由rpaA 基因插入突變，影響「晝夜節律調控× 光保護色素系統強化 × 能量分配權衡」三者共同驅動的整體適應轉型。研究結果深化了對藍綠菌高光適應機制的理解，並為利用晝夜節律因子提升光合生物耐逆境能力提供理論依據。 ;This study investigates the evolution of high-light (HL) tolerance and photoprotective strategies in Cyanobacterium aponinum under extreme HL stress. Wild-type cells exhibited growth arrest and pigment degradation under 1200 μmol photons m⁻² s⁻¹, with only a small fraction surviving and resuming proliferation, highlighting rapid adaptive potential in their photosynthetic and regulatory networks. Selected HL-tolerant mutants maintained stable photosystem function, enhanced energy dissipation, and antioxidative capacity, while sustaining growth rates comparable to the wild type under continuous light, indicating that HL adaptation occurred without significant growth costs. Whole-genome analysis revealed few accumulated mutations in HL-tolerant strains. In particular, the AL20115HL-2 mutant harbored a 9-bp insertion in the rpaA gene, resulting in three amino acid insertions. RpaA, a key output transcription factor of the circadian clock, regulates photosynthesis, carbon metabolism, and repair pathways. Structural modeling suggests that this insertion may subtly alter RpaA dimerization and DNA-binding affinity, thereby modulating transcriptional outputs of hundreds of downstream genes and enabling rapid adaptation to high light stress. Pigment analysis showed a significant increase in carotenoids and a decrease in chlorophyll a, reflecting typical photoprotective reprogramming. This "reduced light harvesting + enhanced energy dissipation" strategy effectively mitigates photodamage. Although growth under light-dark cycles was slower, likely due to altered circadian output from the rpaA insertion, the mutant retained a clear survival advantage under HL conditions. In summary, the high-light-tolerant phenotype of C. aponinum AL20115HLr-2 is likely driven by an insertional mutation in the rpaA gene, which induces an integrated adaptive transition jointly mediated by circadian regulation, enhancement of photoprotective pigment systems, and trade-offs in energy allocation. These findings deepen our understanding of high-light adaptation mechanisms in cyanobacteria and provide a theoretical framework for exploiting circadian regulatory factors to enhance stress tolerance in photosynthetic organisms.

解析阿拉伯芥CYP65蛋白質參與HIT4調控熱誘導染色質中心重塑機制;Investigating the role of Arabidopsis CYP65 in HIT4-regulated heat-induced chromocenter remodeling

Fri, 06 Mar 2026 10:24:23 GMT

title: 解析阿拉伯芥CYP65蛋白質參與HIT4調控熱誘導染色質中心重塑機制;Investigating the role of Arabidopsis CYP65 in HIT4-regulated heat-induced chromocenter remodeling abstract: 高溫逆境對植物生長與存活造成嚴重威脅，而染色質結構的動態調控被認為是植物耐熱性的重要分子機制。本研究聚焦於阿拉伯芥蛋白 HIT4 在熱逆境下的功能及其相關調控路徑，發現 HIT4 可促使 CYP65 正確定位至染色質中心，兩者在染色質中心呈現高度的共定位，並具有蛋白交互作用。細胞定位分析顯示，在常溫條件下，HIT4 主要集中於染色質中心，而 CYP65 分布於細胞核內；經熱逆境處理後，CYP65 受熱誘導重新定位至染色質中心，與 HIT4 的定位相同。而植物熱逆境表現型結果顯示，cyp65 突變株在長時間高溫處理下與 hit4 具有同樣對熱敏感的表現，且 cyp65經熱逆境處理後染色質中心鬆散不完全，呈現多個密集小點的中間型態；相比之下，野生型植株的染色質中心可完全鬆散，而 hit4 突變株則維持高度凝集。進一步觀察發現，在 hit4 突變背景中，CYP65 無法被熱誘導至染色質中心，表明其定位依賴 HIT4 的協助。綜合結果顯示，CYP65 為 HIT4 調控熱逆境下染色質中心結構重塑的重要因子。本研究揭示了 HIT4 與 CYP65 在植物熱逆境應答中調控染色質結構的新機制，為理解植物耐熱性的表觀遺傳調控提供了新的研究方向。;Heat stress poses a severe threat to plant growth and survival, and dynamic regulation of chromatin structure is considered an important molecular mechanism underlying plant thermotolerance. This study focused on the function of the Arabidopsis protein HIT4 under heat stress and its associated regulatory pathway, and found that HIT4 facilitates the proper localization of CYP65 to chromocenters, with the two proteins exhibiting high colocalization and direct protein–protein interaction. Subcellular localization analyses showed that under normal conditions, HIT4 is mainly concentrated at chromocenters, whereas CYP65 is distributed throughout the nucleus; following heat stress, CYP65 is specifically recruited to chromocenters, exhibiting the same localization as HIT4. Phenotypic analyses under heat stress revealed that cyp65 mutants display heat sensitivity similar to that of hit4, and that mutant chromocenters are incompletely decondensed, forming multiple dense foci, whereas in wild-type plants, chromocenters are fully decondensed, and in hit4 mutants, they remain highly condensed. Further observation indicated that in the hit4 mutant background, CYP65 fails to be recruited to chromocenters upon heat stress, indicating that its localization depends on HIT4. Collectively, these results demonstrate that CYP65 is a critical component in HIT4-mediated chromocenter remodeling under heat stress. This study reveals a novel mechanism by which HIT4 and CYP65 regulate chromatin structure in plant heat stress responses, providing new insights into the epigenetic regulation of plant thermotolerance.

Analysis of the feasibility of enhancing plant thermotolerance through substitution of farnesylation on AtJ3 with geranylgeranylation and establishment of a plant protein geranylgeranylation platform in E. coli.

Fri, 06 Mar 2026 10:24:01 GMT

title: Analysis of the feasibility of enhancing plant thermotolerance through substitution of farnesylation on AtJ3 with geranylgeranylation and establishment of a plant protein geranylgeranylation platform in E. coli. abstract: 蛋白質異戊二烯化（protein prenylation），包括法尼脂化（farnesylation）與香葉基化（geranylgeranylation），是一類重要的蛋白質脂質修飾機制。含有 CaaX 端序列的蛋白質會由異二聚酶組成的法尼基轉移酶（PFT）或香葉基轉移酶（PGGT-I）進行修飾，而此類修飾在植物中對蛋白質細胞內的定位、穩定性與功能，尤其在逆境反應中，具有關鍵調控作用。在 Arabidopsis thaliana 中，HSP40 蛋白 AtJ3 需要接受法尼脂化，才可以與HSP70-4於stress granule協同運作並提供耐熱性；然而，香葉基化是否能在熱耐受性方面取代法尼脂化，仍未知曉。在本研究中，我們於 Escherichia coli 中共表現 AtPGGTα、AtPGGTβ 以及 AtGGPPS2，提供功能性的香葉基焦磷酸（GGPP）以建立植物蛋白質香葉基化系統。利用此系統，我們確認三種CaaX修改過的 AtJ3（AtJ3CHLF、AtJ3CSFL、AtJ3CAFL）能被AtPGGT辨識並進行修飾。此外，透過先前研究於E. coli建立的植物法尼脂化辨識系統，我們也發現 AtJ3CHLF 與 AtJ3CSFL可在 E. coli 中接受法尼脂化，顯示其具有被法尼脂化也具有被香葉基化修飾的能力，然而AtJ3CAFL則只具有被香葉基化修飾的能力。將這些修改的 AtJ3 的基因導入 Arabidopsis j3 突變株後所得之轉殖株，在耐熱試驗中皆顯示三種轉殖株熱耐受性皆弱於野生型 AtJ3，顯示香葉基化在維持 AtJ3 所介導的耐熱功能上效果較差。也因此影響了轉殖株的耐熱能力。綜合而言，本研究揭示 CaaX 序列組成會決定 AtJ3 所接受的異戊二烯修飾類型，並深刻影響其熱耐受性。我們建立的細菌系統提供一個研究植物蛋白質香葉基化的新平台，同時也提出明確證據指出：相較於香葉基化，法尼脂化對植物獲得最佳熱耐受性更為關鍵。 ;Protein prenylation, including farnesylation and geranylgeranylation. Protein with N-terminal CaaX motif is catalyzed by a heterodimeric protein farnesyltransferase (PFT), geranylgeranyltransferase (PGGT-I). It is a crucial post-translational lipid modification that regulates protein localization, stability, and function in plant stress responses. In Arabidopsis thaliana, the HSP40 chaperone AtJ3 requires farnesylation to cooperate with HSP70-4 within stress granules and thereby confer thermotolerance. However, whether geranylgeranylation can functionally substitute for farnesylation for heat tolerance remains unknown. In this study, we co-expressed AtPGGTα, AtPGGTβ, and AtGGPPS2 in Escherichia coli to generate a functional supply of geranylgeranyl pyrophosphate (GGPP) and thereby establish a plant protein geranylgeranylation system. Using this platform, we demonstrated that three CaaX-modified AtJ3 variants (AtJ3CHLF, AtJ3CSFL, and AtJ3CAFL) were recognized and modified by AtPGGT. In addition, by employing a previously established plant protein farnesylation system in E. coli, we found that AtJ3CHLF and AtJ3CSFL, but not AtJ3CAFL, could also undergo farnesylation, indicating alternative prenylation potential for AtJ3CHLF and AtJ3CSFL, whereas AtJ3CAFL is restricted to geranylgeranylation. Transgenic Arabidopsis j3 mutant lines expressing full-genomic engineered AtJ3 (AtJ3CHLF, AtJ3CSFL, and AtJ3CAFL) exhibited reduced thermotolerance compared with wild-type AtJ3, suggesting that geranylgeranylation is less effective than farnesylation in maintaining AtJ3-mediated heat protection. Additional assays revealed that AtJ3CHLF and AtJ3CSFL, but not AtJ3CAFL, could also be farnesylated in E. coli, indicating AtJ3CHLF and AtJ3CSFL have ability of both farnesylation and geranylgeranylation, AtJ3CAFL have ability of geranylgeranylation. These findings demonstrate that CaaX motif composition critically determines the type of prenylation and its functional consequences. This study establishes a bacterial platform for analyzing plant protein geranylgeranylation and provides mechanistic evidence that farnesylation, rather than geranylgeranylation, is essential for optimal thermotolerance in plants.

y-TuRC 調控 KIF2A 所引導的微管解聚合作用;y-TuRC Regulates Kinesin Superfamily Protein 2A(Kif2A)-Dependent Microtubule Depolymerizing Activity

Fri, 17 Oct 2025 05:51:49 GMT

title: y-TuRC 調控 KIF2A 所引導的微管解聚合作用;y-TuRC Regulates Kinesin Superfamily Protein 2A(Kif2A)-Dependent Microtubule Depolymerizing Activity abstract: 微管（Microtubules）是細胞骨架家族中的一員，由 a/b-微管蛋白（a/b-tubulin）異二聚體組成的高度動態聚合之絲狀結構。微管能夠在聚合 (polymerization) 與解聚（depolymerization）之間展現動態變化，此動態對於形成細胞內不同微管網路與微管結構至關重要(例如:紡錘體與纖毛) ，進一步調控許多的細胞功能。而許多微管作用蛋白（microtubule-associated proteins），包括驅動蛋白（kinesin）馬達，能夠調控微管的生長與收縮。本研究中 Kif2A 是眾多驅動蛋白家族中kinesin-13 家族的一員，雖然也屬於驅動蛋白，但其主要的活性為促進微管解聚。我們先前的研究發現了一個依賴由八個蛋白質所組成的 a/b-微管環狀複合體（y-TuRC）的分子機制，此機制可招集 Kif2A 至纖毛的基體（basal body），並促進纖毛分解（cilia disassembly），調控纖毛形成在細胞週期中的形成時機。我們的研究進一步顯示，a/b-微管環狀複合體透過其中兩個次單元蛋白 GCP2 及其結合蛋白 Mzt2 專一性地招集 Kif2A到纖毛之基體。在本論文研究中，我們計畫依據先前的研究結果，進一步利用生物物理學、生物化學及結構生物學的方法，探討 Kif2A 如何與 a/b-微管環狀複合體中的次單元蛋白 GCP2 與Mzt2 結合。以及此結合是否能夠調控 Kif2A 的微管解聚活性。未來，我們希望進一步探討 a/b-微管環狀複合體與 Kif2A 在調控微管解聚的分子機制，以控制不同微管結構的形成, 例如:纖毛解離過程。;Microtubules, composed of a/b-tubulin heterodimers, are highly dynamic polymers that undergo continuous phases of polymerization and depolymerization. This dynamic behavior is essential for the assembly of diverse microtubule-based structures—such as the mitotic spindle and cilia—and supports a broad range of cellular processes. Many microtubule-associated proteins (MAPs), including kinesin motors, modulate microtubule dynamics by promoting either growth or shrinkage. Kinesin-13 family members, such as Kif2A, are specialized microtubule depolymerases. In this study, we examine how Kif2A activity is regulated by !- tubulin ring complex (y-TuRC). Our previous study uncovered a y-TuRC-dependent pathway that recruits Kif2A to the basal body, promotes cilia disassembly and prevents unscheduled cilia formation. Our mechanistic analyses showed that y-TuRC specifically recruits Kif2A via the GCP2 subunit and its binding partner Mzt2. Here we propose to apply biophysics and biochemistry approaches to examine how Kif2A binds to y-TuRC subunits, the GCP2/Mzt2 complex, and to determine whether this interaction modulates Kif2A’s microtubule depolymerizing activity. Ultimately, we would like to investigate how y-TuRC and Kif2A cooperatively regulate microtubule depolymerization to drive the remodeling of microtubule-based structures, such as during cilia disassembly