FIN219與FHY1在阿拉伯芥遠紅光訊息傳遞中的調控關係之研究

Study of Regulatory Relationship between FIN219 and FHY1 in PHYA-mediated Far-red Light Signaling Pathway in Arabidopsis

李森畫(Sen-Hua Li)

指導教授 ： 謝旭亮

國立臺灣大學/生命科學院/植物科學研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.00083

摘要

在阿拉伯芥中，光敏素A（phytochrome A, phyA）是感受遠紅光的光接受體，並且調節各種受到遠紅光引發的光型態發生反應；先前的研究已發現了多種phyA訊息調控因子，例如FIN219 (far-red insensitive 219)以及 FHY1 (far-red elongated hypocoty 1)，然而各個成員之間的調控機制尚不明瞭。已知FIN219是phyA調控的遠紅光訊息傳遞中的傳訊因子之一，同時FHY1基因也專一參與在遠紅光訊息傳遞的下游，前人研究發現兩者表現出非等位非互補性(nonallelic noncomplementation)的遺傳關係，暗示FIN219與 FHY1可能參與在同一個訊息傳遞中。將fin219 null突變株與fhy1-T突變株進行雜交測試時，發現F3子代的同型合子雙突變株在強遠紅光中呈現像fhy1-T的外表型，弱遠紅光下則是對於光照有更不敏感的長下胚軸外表型，比個別親代突變株的下胚軸還長，推測兩者蛋白質之間可能會有結合作用共同去調控phyA調控的植物生長發育。因此分別利用酵母菌雙雜交系統以及雙分子螢光互補系統測試兩蛋白質之間的結合能力，證實FHY1與FIN219發生交互作用的位置主要在細胞核，或者同時位在細胞質和細胞核中，並且證實FHY1以N端的部分與FIN219發生交互作用。另外，分別利用北方墨點法和西方墨點法比較野生型、fin219 null突變株及fhy1-T突變株中，各別FHY1和FIN219在基因層次以及蛋白質層次的表現情形，結果顯示FIN219會負調控FHY1基因表現量，但會幫助FHY1在細胞中的累積；而FHY1則是會些微負調控FIN219，說明兩者之間可能存在一種負向回饋的調控關係。進一步利用轉殖株外表型的檢定，發現大量表現FHY1及FIN219皆會部份回復個別fin219 null和fhy1-T突變株的外表型。總結上述結果，認為FIN219與FHY1在遠紅光的訊息傳遞路徑中，具有結合能力共同正向調控阿拉伯芥中的光型態發生，然而植物中FIN219和FHY1之間交互作用的生理意義，以及進一步會影響到哪些調控機制則尚待更多實驗研究來解開這個謎題。

關鍵字

光敏素訊息調控 ； 遠紅光 ； 雙分子螢光互補系統 ； 酵母菌雜合法 ； 非遺傳非互補

並列摘要

Far-red insensitive 219 (FIN219) was previously shown to be involved in phytochrome A-mediated far-red (FR) light signaling in Arabidopsis. Far-red Elongated Hypocoty l (FHY1) is a positive regulator and required for FR-regulated nuclear accumulation of phyA. Previous study indicated that the fin219/fhy1 transheterozygote exhibited a long hypocotyl phenotype in FR similar to their homozygous parental mutants, suggesting that fin219 and fhy1 mutants are nonallelic noncomplementary. However the molecular mechanism underlying the genetic interaction of FIN219 and FHY1 remains largely unknown. We use molecular genetics and cell biology approaches to understand their interaction and expression patterns in Arabidopsis. Firstly, we crossed the T-DNA insertion mutants of fin219 null and fhy1-T to generate a homozygous double mutant, which further exhibited an enhanced far-red insensitive phenotype compared to its respective parental mutants in weak FR, but same as fhy1-T in strong FR. In phenotypic analysis of transgenic plants, we found that over-expression of FIN219 or FHY1 in fhy1-1 or fin219 null respectively can partially rescue respective long-hypocotyl phenotype. Hence, we hypothesize that FIN219 may interact with FHY1. By using yeast two-hybrid and bimolecular fluorescence complementation approaches, we demonstrated that they interacted with each other in the nuclei only, and also in both the nuclei and the cytoplasms, and further found that the N-terminal region of FHY1 is responsible for the interaction with FIN219. In subcellular localization studies, FIN219-YFP displayed different patterns in the fhy1-T protoplasts compared with that in wildtype, whereas FHY1-CFP was localized with the same patterns in the protoplasts of different mutant or transgenic plants. Further expression studies revealed that FIN219 negatively regulated FHY1 transcripts and positively FHY1 protein in both darkness and FR; whereas, FHY1 slightly negatively regulated FIN219 protein, instead of FIN219 transcript levels in darkness and FR. Taken together, these data indicate that FIN219 and FHY1 interacted with each other and were positively involved in phyA-mediated photomorphogenesis in Arabidopsis.

並列關鍵字

FHY1 ； FIN219 ； phyA ； FR ； BiFC ； yeast two-hybrid ； non-allelic non-complimentation

參考文獻

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