謝旭亮臺灣大學：植物科學研究所李柏均Lee, Pai-ChunPai-ChunLee2007-11-272018-07-062007-11-272018-07-062004http://ntur.lib.ntu.edu.tw//handle/246246/57992中文摘要 光線對植物的生長發育有著密不可分的關係，而目前已知植物用來接收光的接受體有光敏素(phytochrome)、cryptochromes、phototropins，且各種光接受體會利用不同的下游傳遞因子來幫助訊息的傳導。其中，FIN219基因能表現出一個575胺基酸的GH3-like蛋白質，並參與遠紅光下游的訊息傳遞。有趣的是，在阿拉伯芥基因體中存在著至少20個GH3-like的基因，它們之間在胺基酸序列上具有35%~47%的相同性，也存在著一些很保守(conserved)的區域，但是它們的功能尚不清楚。 為了更明白阿拉伯芥中除了FIN219以外其他的GH3-like基因群是否也參與光訊息傳遞，我們利用T-DNA 插入的阿拉伯芥突變體及許多已知的光訊息調控的阿拉伯芥突變體為材料，在黑暗、藍光、遠紅光、紅光的光源下分析各種GH3 T-DNA 插入突變體的下胚軸延長情形與野生型的差異。結果顯示所有的T-DNA插入突變體在紅光與遠紅光下均有較野生型明顯長的下胚軸長度，證實阿拉伯芥中大部分GH3-like基因的確參與紅光及遠紅光所誘導的下胚軸延長過程。另外，利用RT-PCR的方法在白光、黑暗、遠紅光下分析不同光訊息突變體中GH3-like基因群的表現情形，結果顯示許多光接受體的突變體均能影響GH3-like基因的表現，其中FIN219、GH3-7和GH3-10可能同時參與兩種以上的光訊息傳遞路徑且在遠紅光中大部分GH3-like基因均受phyA調控，COP1能抑制部分GH3-like基因的表現。除此之外，由於GH3-like基因群中有一個已知會受遠紅光誘導而大量表現的基因GH3-10，所以我利用大量表現與反譯股抑制GH3-10基因表現(Anti-sense)於阿拉伯芥野生型以及幾種突變體中去分析GH3-10的功能，同時也針對GH3-10基因的N端進行轉殖分析；另外，還用基因槍打入法研究GH3-10在細胞層次的座落位置，並以雜交測試法來探討GH3-10與FIN219之間的關係。結果顯示不論抑制或大量表現GH3-10基因於野生型的轉殖株中均造成轉殖株在紅光與遠紅光中表現出較野生型長的下胚軸，這暗示著GH3-10同時參與著紅光及遠紅光的訊息傳遞路徑且可能以形成複合體(complex)的方式行使其功能，所以具有一固定表現劑量(dosage)的需要。另外也發現，只有在正常的phyA、phyB存在的情況下GH3-10才能正常行使其功能，且單獨表現GH3-10的N端232個胺機酸於野生型中均不會影響轉殖株幼苗在各種光源下的外表型，這顯示GH3-10的C端可能在感受光訊息方面具有重要的功能。而用基因槍的方法將GH3-10打入洋蔥的表皮細胞中發現GH3-10主要表現於細胞質中並且它的細胞內座落位置不會受到光的影響而改變。最後由雜交測試顯示GH-10與FIN219具有非等位非互補(Nonallelic noncomplementation)的遺傳特性，暗示兩者可能直接相互作用或作用在phyA下游具有一平行且分岔的調控關係。Abstract Light is one of the most important environmental factors that affect the growth of plants. Three types of photoreceptors identified so far are phytochromes, cryptochromes, and phototropins. Each photoreceptor uses different downstream signal transduction components to transmit light signals. One of these components, identified as FIN219 gene, is involved in phytochrome A-mediated signaling pathway and encodes a GH3-like protein with 575 amino acids. Intriguingly, there are at least 20 GH3-like genes present in Arabidopsis genome. Sequence comparison among them indicates that there is a 35%~47% identity at amino acid level, in which some conserved regions are also found. However, their functions remain unknown. To understand further to see if other GH3-like genes in addition to FIN219 are also involved in light signaling pathway, T-DNA insertion lines and other light signaling mutants in Arabidopsis were used as samples to analyze the possible phenotypes and regulatory relationship under dark, blue, far-red, and red light. The results showed that the hypocotyl lengths of all T-DNA insertion lines are significantly longer than wild type under red and far-red light conditions. This indicated that most of the GH3-like genes are involved in red- and far-red-mediated inhibition of hypocotyl elongation in Arabidopsis. Besides, RT-PCR was ultilized to investigate the expression patterns of different GH3-like genes in different light signaling mutants under dark, white and far-red light. The results indicated that many photoreceptor mutants influence the expression of GH3-like genes. In particular, FIN219, GH3-7 and GH3-10 may be involved in more than one light signaling pathways. COP1 can repress gene expression of some of the GH3-like genes under far-red light. Furthermore, since it was reported that one of the GH3-like genes, GH3-10, can be up-regulated under far-red light and this induction was abolished in phyA mutant. GH3-10 was also most close to FIN219 at amino acid level, leading us to investigate the actual function of GH3-10 in light signaling. The transgenic plants overexpressing and antisensing the GH3-10 in wild type displayed a longer hypocotyl phenotype in red and far-red light than that in wild-type. This implies that GH3-10 participates in both red and far-red light signal transduction pathways and may form a functional complex with a dosage effect to carry out its physiological role in Arabidopsis. Meanwhile, it was also found that GH3-10 was functional only in the presence of phyA and phyB. However, the transgenic plants overexpressing the N-terminal 232 amino-acid portion of GH3-10 did not show any obvious phenotype under all light conditions, implying that the C-terminus of GH3-10 may play more important roles to trigger light signaling. Expression of GH3-10 protein in onion cells using particle bombardment showed that it was localized in cytosol and this subcellular localization is light-independent. At last, the crossing test between GH3-10 and FIN219 suggested that both are nonallelic noncomplementational genetic relationship and may interact with each other or work in parallel with each other in phyA-mediated signal transduction pathway.目錄 中文摘要………………………………………………………………………….…I 英文摘要…………………………………………………………………………...III 第一章 前言 一、 導論……………………………………………………………..…1 二、 植物的光訊息傳遞…..…………………………….……. …….…1 三、 Auxin所影響的植物生長與基因表現………………………….10 四、 研究目標…………………………………………………………12 第二章 材料與方法 一、 植物材料與生長條件……………………………………………….13 二、 GH3-like gene clones……………………………………….……….14 三、 GH3-10基因construct的構築…………………………….……….15 四、 基因轉殖與轉殖株篩選…………………………...……….……….16 五、 RNA的粹取與RNA表現量分析…………………..……….……….16 六、 西方式點墨分析(Western blot)………………………………...…...17 七、 細胞層次座落位置與基因槍法…………………………………….18 八、 雜交測試…………………………………………………………….18 第三章 結果 一、GH3-like基因均具有一coiled-coil domain，且在promoter區域 含有許多可受光調控的cis-elements在…………...….….……19 二、所有GH3-like基因的clones…………….………………….………..……..20 三、T-DNA插入GH3-like基因的阿拉伯芥轉殖株幼苗在紅光與遠紅光下具 有比野生型長的下胚軸外表型……………………..………………..…..20 四、GH3-like基因的T-DNA插入突變體幼苗的外表型能直接反應遠紅光強 度……………………………………….……………………………….…22 五、FIN219與GH3-7受到多種光接受體與下游光訊息傳遞者的調控，並且遠紅光下GH3-like基因在phyA突變體中表現量均有降低，而在cop1-4中則部分GH3-like基因表現會上升…………………………….…..…..22 六、表現GH3-10基因的反譯股(Anti-sense)於野生型阿拉伯芥中造成轉殖株 幼苗在紅光與遠紅光中下胚軸增長……...………….…………………..24 七、大量表現GH3-10基因於野生型阿拉伯芥中仍造成轉殖株在紅光與遠紅光下具有比野生型長的下胚軸………………………...………………...25 八、抑制與大量表現GH3-10基因於phyA、phyB、fin219阿拉伯芥突變體中， 只有在fin219突變體中抑制GH3-10基因表現時有更長的下胚軸外表型 出現………………………..…………………………………….…….…..27 九、大量表現GH3-10的N端於野生型阿拉伯芥中並沒有新的外表型出現，而反譯股抑制GH3-10 N端的轉植株則具有較長的下軸………….…..28 十、GH3-10主要表現於細胞質中並且不會受到光線影響其蛋白質座落位 置……….………………………….……………………..………………..29 十一、fin219突變體與GH3-10 T-DNA插入突變體雜交後的F1子代，其下胚體長度較fin219突變體稍短…………………………..………………29 第四章 討論 一、GH3-like基因可以受到光的調控，並且可能與其他蛋白質產生交互作用………..……………………………………………………..….……….52 二、不同GH3-like基因於野生型阿拉伯芥中表現程度似乎不盡相同，並且不一定會受到auxin所誘導………………………………….…………..53 三、GH3-like基因家族在阿拉伯芥感受紅光與遠紅光的訊息傳遞路徑中扮演著重要角色……………………………………………………………….54 四、GH3-like基因家族在白光與黑暗中受到光接受體與幾種下游基因調控的表現情形不完全相同，且在遠紅光下COP1似乎會抑制部分GH3-like基因的表現…………………………………………………….…………55 五、表現GH3-10基因的反譯股(Anti-sense)於野生型阿拉伯芥中造成轉殖株對紅光與遠紅光的敏感度下降…….……………………………………57 六、阿拉伯芥中GH3-10蛋白質可能需要與其他蛋白質結合成一聚合物的形 式才能行使其功能……………………….………………………………58 七、GH3-10與FIN219間具有非等位非互補性的遺傳特性暗示兩基因於phyA下游彼此間具有平行且分岔的調控關係………..………………59 八、GH3-10真正具有關鍵性功能的區域可能在C端……………………60 九、GH3-10主要表現於細胞質中並且不會受到光線影響其蛋白質座落位置……………………………………………………….……………….61 十、大量表現GH3-10與大量表現DFL2時造成轉殖株外表型的差異….62 參考文獻……………………………………….………………………….….…….63 附錄一、光訊息傳遞路徑…………………………………………………………73 附錄二、實驗中所用表現載體……………………………………………………74 附錄三、阿拉伯芥之無菌栽培…………………………………….………..…….76 附錄四、總RNA的抽取………………………………………………………….77 附錄五、Quantitative RT-PCR……………………………….….……………….78 附錄六、Gene cloning…………………………….….……………………..……..81 附錄七、Genomic DNA粹取……….….……………………………………..…..84 附錄八、基因槍法（particle bombardment）…………………………………..85 附錄九、表現載體之構築….….…………………………………………….……..88 附錄十、阿拉伯芥之轉殖及篩選………………………………………….………91 附錄十一、蛋白質電泳與西方點墨法(Western blot) …………………….……...94en-US光訊息GH3other