- 學位論文
利用基因剃除技術研究藍綠菌Synechococcus sp. PCC 7335中可吸收 遠紅光的光系統一
Mutagenesis study of the Photosystem I that absorbs far-red light in a cyanobacterium Synechococcus sp. PCC 7335
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摘要
大部分的光合生物可以利用波長 400-700 奈米的可見光能源。然而，有一些品種的 藍綠菌除了可以利用 400-700 nm 波長的能源外，也可以利用遠紅光 (波長 700-800 奈米)。當生活在可見光較少且遠紅光較多的環境時，它們可以行遠紅光轉換作用 來適應環境。經由遠紅光轉換，這些藍綠菌會產生葉綠素 d、f，以及表現遠紅光基 因簇中的 20 個基因來重組光系統一、二以及藻膽蛋白體。近期冷凍電子顯微鏡解 出的藍綠菌遠紅光下的光系統一結構，揭露了幾個可能的葉綠素 f 結合位點。然而， 葉綠素 f 以及葉綠素 a 結構上非常相似，因此以目前的技術要能夠直接區分出兩者 具有非常大的挑戰性，在一些解析度比較侷限的區域上更是不容易確定。並且，每 個葉綠素 f 在光系統一中所扮演的角色也不清楚。為了彌補結構研究上的不足，我 們選定了在遠紅光光系統一中坐落在可能葉綠素 f 結合位點附近的四個基因(psaF2, I2, J2, and L2)來進行基因剃除。結果顯示 psaL2-突變株不但在遠紅光下長得比較 慢，而且同時他們的葉綠素 f : a 比例也降低了。此外我們也發現缺少 PsaF2 和 PsaJ2 並不會影響到光系統一的三聚體結構，但卻會改變光譜特性。再者，結合先前的結 構研究以及以高效液相層析測量得出的結果－在遠紅光下突變株 psaJ2-光系統 I 中 的葉綠素 f 含量比率和野生株以及突變株 psaF2-的光系統 I 中的含量來的低可以推 斷出，在蛋白為單元體 PsaJ2 附近有一個葉綠素 f 的結合位。
並列摘要
Most oxygenic phototrophs use photosynthetically active radiation (PAR; wavelengths = 400-700 nm) for photosynthesis. However, some special cyanobacteria can use PAR and far-red light (FRL, wavelengths = 700-800 nm). When these cyanobacteria grow in FRLenriched environments, they perform far-red light photoacclimation (FaRLiP) to harvest FRL. While performing FaRLiP, they synthesize chlorophyll d (Chl d) and chlorophyll f (Chl f) and express 20 genes in the FaRLiP gene clusters to remodel their photosystem I (PSI), photosystem II, and also phycobilisome. Recent PSI structural cryogenic electron microscopy (cryo-EM) studies identified several possible Chl f binding sites in the FRLremodeled PSI. However, the structure of Chl f and Chl a are too similar, and in some regions with lower resolution, they are hard to differentiate under the resolution of cryoEM. In addition, the function of each Chl f molecule in PSI is still unknown. To complement the deficiency of cryo-EM, we individually used conjugation and homologous recombination to knockout the four PSI subunits (PsaF2, I2, J2, and L2) in the FaRLiP gene cluster in Synechococcus sp. PCC 7335. These four subunits are near the proposed binding sites in the cryo-EM structure. We found that the psaI2- and psaL2- mutant grows slower than the wild type under FRL and decreases the Chl f: a ratio. Also, the PSI purification result shows that the lack of PsaF2 or PsaJ2 does not affect trimerization but changes the spectral properties of the complex. Moreover, combined with previous structural studies and the HPLC analysis that the Chl f molecules ratio is decreased in the purified FRL PSI of psaJ2-, there is a Chl f binding site near PsaJ2.
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