Investigation of the Functional Relationships between FIN219 and FHY1 Genes.

植物光敏素是一種光接受體，可以感受光照而調節植物生長與發育。在阿拉伯芥中，光敏素A（PHY A）是主要感受遠紅光並調節各種受到遠紅光引發的反應。先前的研究發現了多種PHYA訊息調控因子，例如FIN219 (far-red insensitive 219)以及 FHY1 (far-red elongated hypocoty 1)。已知FIN219基因是專一作用在PHYA調控的遠紅光訊息傳遞的傳訊因子之一，FHY1基因也是參與在遠紅光訊息傳遞的下游。將fin219突變株與fhy1突變株進行雜交測試時，發現F1子代在遠紅光下有長下胚軸外表型，且比個別突變株在heterozygote時的下胚軸長，顯示兩者具有非等位非互補（nonallelic-noncomplementation）的遺傳調控關係，很可能共同參與訊息傳遞的途徑，甚至兩者蛋白質會有結合作用。因此利用酵母菌雙雜交系統測試兩蛋白質之間的結合能力。結果發現FHY1可以與FIN219 C端有結合能力。 當FIN219載體轉殖入fhy1突變株中，遠紅光中生長的轉殖株幼苗，會有比fhy1突變株更短的下胚軸長度，說明fhy1突變株對於遠紅光的感受能力變得比較強；而大量表現FHY1在fin219突變株中，同樣使得轉殖株的下胚軸長度比fin219突變株還要短。此外，北方墨點法的分析結果指出，黑暗中生長的fin219突變株中的FHY1基因表現增加；而黑暗中生長的fhy1突變株中的FIN219基因表現量也會上升。 總結上述結果，認為FIN219與FHY1分處訊息傳遞的兩個平行途徑上，並且具有結合能力而共同調控遠紅光的訊息傳遞。Phytochromes are plant photoreceptors that regulate plant growth and development in response to the light environment. In Arabidopsis, phytochrome A (phyA) is the primary photoreceptor responsible for perceiving and mediating various responses to far-red light. Previous studies have identified multiple phyA signaling components, such as FIN219 (far-red insensitive 219) and FHY1 (far-red elongated hypocoty 1). FHY1 encodes a product (FHY1) that specifically transduces signals downstream of the far-red (FR) light-responsive phytochrome A (PHYA) photoreceptor. FIN219 is also involved in phytochrome A signaling pathways. Complementation test between fin219 and fhy1 show a nonallelic noncomplementation relationship, indicating possible genetic interactions between these two proteins. Hence, we carry out the yeast 2-hybrid assay to investigate this possibility. The results show that FHY1 protein can interact with FIN219 C-terminus. As well, when the FIN219 overexpression construct was introduced into the fhy1 mutant, the transgenic seedlings show shorter hypocotyls than fhy1 under FR condition, which indicates that the fhy1 mutant gains more sensitivity to the perception of FR light. Moreover, the transgenic seedlings overexpressing the FHY1 in the fin219 mutants also result in a shorter hypocotyl phenotype when compared to that of fin219. In addition, Northern analysis revealed that FHY1 transcript levels arise in fin219 mutants grown in darkness; at the same time, FIN219 transcript levels increase in fhy1 mutant as well. In summary, the results indicate that FIN219 and FHY1 coordinate with each other by direct interaction and response to FR light.中文摘要.....................................................................................................I Abstract....................................................................................................II 縮寫表......................................................................................................III 第一章 前言...........................................................................................1 一、導論...............................................................................................................1 二、植物的光訊息調控.......................................................................................1 1. Phytochromes (PHYs)光敏素.................................................................2 2.Cryptochrome (CRYs)隱花色素..............................................................5 三.光訊息傳遞因子..............................................................................................8 1. FIN219基因...........................................................................................10 2.FHY1基因...............................................................................................10 第二章 材料與方法.............................................................................13 一、載體的構築(Plasmid Construction)..........................................................13 二、植物材料與生長條件.................................................................................17 三、基因轉殖與轉殖株篩選.............................................................................18 四、雜交測試.....................................................................................................19 五、北方式點墨分析(Northern blot)...............................................................19 六、西方式點墨分析(Western blot).................................................................20 第三章 結果........................................................................................22 一、將FIN219大量表現在fhy1突變株中，增進fhy1突變株對光的敏感性..............................................................................................................................22 二、大量表現FHY1在fin219突變株中，增進fin219突變株對光的敏感性..............................................................................................................................23 三、FHY1與FIN219蛋白質的C端具有交互作用.........................................24 四、COP1與FIN219蛋白質的C端具有交互作用.........................................25 五、FHY1大量表現在cop1-6突變株並不影響其性狀..................................26 六、fhy1突變株與cop1-6突變株雜交後的子代F1，表現出與fhy1突變株、cop1-6突變株不同的性狀.................................................................................26 七、以北方墨點法（Northern bolt）分析FHY1與FIN219基因表現........27 圖1、大量表現FIN219在fhy1突變株中的轉殖株，在遠紅光中的外表型30 圖2、以北方墨點法分析FIN219大量表現在fhy1突變株中時，FIN219與FHY1的基因表現量。......................................................................................32 圖3、以西方墨點法分析FIN219大量表現在fhy1突變株中時，FIN219蛋白質表現量。.........................................................................................................33 圖4、大量表現FHY1在fin219突變株中的轉殖株，在遠紅光中的生長情形。..............................................................................................................................35 圖5、酵母菌雙雜交質體構築圖（一）..........................................................36 圖6、以酵母菌雙雜交系統檢測FHY1與FIN219的結合能力.....................38 圖7、以酵母菌雙雜交系統檢測COP1不同片段與FIN219的結合能力.....40 圖8、FHY1大量表現在cop1-6突變體中的轉殖株，在遠紅光中及黑暗中的生長情形.............................................................................................................41 圖9、fhy1突變株與cop1-6突變株雜交後的子代F1，在黑暗及遠紅光中生長四天的性狀.........................................................................................................42 圖10、以北方墨點法分析FHY1基因在黑暗中與遠紅光下，不同突變體中的表現......................................................................................................................44 圖11、以北方墨點法分析FIN219基因在黑暗中與遠紅光下，不同突變體中的表現.................................................................................................................45 圖12、以北方墨點法分析不同光照時間處理下，FHY1基因的表現.........46 第四章 討論..........................................................................................47 一、FIN219大量表現在fhy1突變株中，可以改善fhy1突變株對於遠紅光的感受能力.............................................................................................................47 二、FHY1大量表現在fin219突變株中，可以改善fin219突變株對於遠紅光的感受能力.........................................................................................................48 三、酵母菌雙雜交系統的結果說明，FHY1可以與FIN219的C端蛋白質有結合能力.................................................................................................................48 四、COP1與FHY1之間的基因調控關係.......................................................50 五、FHY1與FIN219的基因表現有互相調控的關係....................................51 六、結論.............................................................................................................52 圖13、FIN219與FHY1之間可能的調控關係...............................................53 參考文獻...................................................................................................54 附錄...........................................................................................................6