2020-08-012024-05-17https://scholars.lib.ntu.edu.tw/handle/123456789/687961摘要：有些藍綠菌在可見光不足時能利用遠紅光行放氧光合作用。這個機制稱為遠紅光轉換。遠紅光轉換會產生葉綠素d和葉綠素f，並改裝光合系統一、光合系統二和藻膽蛋白體來吸收遠紅光。這些基因形成一個遠紅光基因簇，他們的表現被光敏素RfpA、RfpC，和轉錄因子RfpB所調控。此計畫第一步是釐清遠紅光在環境中的重要性。目前未知有利用遠紅外光的植物，而利用遠紅光的藍綠菌多為陸生。台灣不同條件的森林、溫泉，和適合藍綠菌生長的溫度與濕度，潛藏著發現更多利用遠紅光機制和物種的可能性。遠紅光基因簇，葉綠素d和葉綠素f 可作為篩選的標記。 可藉由這些標記檢測利用遠紅光藍綠菌的分佈及數量和環境的關係，再進一步純化利用遠紅外光的新種。此外，可利用葉片光譜探測儀在環境中篩選吸收遠紅光的植物，然後分析它們在遠紅光下之生長。第二步是遠紅光轉換的應用性。藍綠菌是生產生質能源或是高價化合物很好的平台，但是藍綠菌中卻沒有一個強力誘導型啟動子。Rfp調控的基因在遠紅光下表現最高增加七萬倍，會是很好的強力誘導型啟動子。可利用染色質免疫沉澱-測序找出RfpB下游的結合序列並設計合成啟動子, 然後在其他藍綠菌應用這套受光調控的強力表現系統。<br> Abstract: Utilization of far-red light (FRL) for oxygenic photosynthesis is important for some cyanobacteria growing in limited accessibility to visible light. Far-Red Light Photoacclimation (FaRLiP) is a photoacclimative process that transforms cyanobacteria to the status that can harvest FRL. This process includes syntheses of two FRL-absorbing chlorophylls (Chl), Chl d and Chl f, and remodeling of photosynthetic apparatus, photosystem I (PSI), photosystem II (PSII), and phycobilisome (PBS) to harvest FRL for photochemistry. The capability for performing FaRLiP comes from genes encoded in a FaRLiP gene cluster, which includes Chl f synthase, paralogous subunits of PSI, PSII, and PBS, and a phytochrome-based transcriptional regulatory cascade (the phytochrome RfpA, the downstream phosphate shutter RfpC, and the transcriptional factor RfpB). There are at least 15 sequenced species across all five taxonomic groups in cyanobacteria are identified containing the FaRLiP gene cluster. The first aim in this proposal is to study the importance of FRL-utilization in photosynthesis in the environment. In addition to FaRLiP, other cyanobacteria and some algae have also evolved strategies independent from FaRLiP to harvest FRL. However, FRL was not identified supporting photosynthesis and growth in plants. Cyanobacteria using FRL are mostly found in terrestrial environments like hot springs, soils, and microbial mats. Therefore, Taiwan is an excellent place to identify unknown mechanisms and species in utilizing FRL because of the various forest systems, hot springs, and the optimal temperature and humidity for cyanobacteria. Using genes in FaRLiP gene cluster, Chl d, and Chl f as markers, the location, distribution, and abundance of FRL-using cyanobacteria in Taiwan can be determined by sample collection, HPLC, and 16s rRNA amplicon sequencing. Samples with potential new species can be subjected to further isolation and purification. Portable Leaf Spectrometer is an accessible method to measure FRL-absorbance of leaves in the field. By comparing spectroscopy of plants species grown under high light and low light area, plants potentially harvest FRL and their distribution can be identified. The growth of the selected species will be evaluated under FRL. The second aim is the application of FaRLiP. Cyanobacteria play excellent roles as cell factories in producing biofuel and valuable compounds. However, there are no strong and precise inducible promoters in controlling gene expression in cyanobacteria. Because genes in FaRLiP cluster is regulated by Rfp, and the highest increase in expression level is 74,500-fold in FRL, the strong and strict Rfp regulatory system can provide strong inducible promoters to express genes of interest in the industry. Furthermore, the expression is regulated by light; therefore, it is reversible and free from external addition of chemicals. By using ChIP-seq, we can identify the essential cis-elements bound by RfpB, the transcription factor in Rfp regulatory system. Based on the information, we can understand which genes are directly controlled by RfpB, design synthetic promoters regulated by FRL, and apply this strong and reversible expression system to other cyanobacteria or plants. Overall, this study will show the importance of FRL in oxygenic photosynthesis and will open up the possibility to use the mechanism of FaRLiP in the application.藍綠菌放氧光合作用遠紅光遠紅光轉換遠紅光基因簇光合系統一光合系統二藻膽蛋白體葉綠素d葉綠素f染色質免疫沉澱-測序合成啟動子高效液相色譜法光譜學cyanobacteriaoxygenic photosynthesisfar-red lightFar-Red Light Photoacclimation (FaRLiP)FaRLiP gene clusterphotosystem Iphotosystem IIphycobilisomechlorophyll dchlorophyll fRfpARfpBRfpCchromatin immunoprecipitation (ChIP)