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National Chung Hsing University Institutional Repository - NCHUIR > 農業暨自然資源學院 > 生物科技學研究所 > 依資料類型分類 > 碩博士論文 >  阿拉伯芥與蕨類GIGANTEA(GI)同源基因之功能性分析顯示其N 端與C 端可能之功能差異

Please use this identifier to cite or link to this item: http://nchuir.lib.nchu.edu.tw/handle/309270000/152591

標題: 阿拉伯芥與蕨類GIGANTEA(GI)同源基因之功能性分析顯示其N 端與C 端可能之功能差異
Characterization and Functional Analysis of GIGANTEA (GI) Orthologues from Arabidopsis and Ferns Reveals the Possibly Functional Diversity for their C- and N-terminal Portion
作者: 沈姿儀
Shen, Tzu-Yi
Contributors: 楊長賢
Chang-Hsien Yang
生物科技學研究所
關鍵字: 開花時間;阿拉伯芥;蕨類
GIGANTEA;flowering time
日期: 2013
Issue Date: 2013-11-18 10:44:27 (UTC+8)
Publisher: 生物科技學研究所
摘要: GIGANTEA (GI) 基因受植物生理時鐘 (circadian clock) 調控,進而促進植物開花。阿拉伯芥中GI 基因突變後會延遲植株開花時間,同時亦對氧化逆境有較高的抗性。為了更深入瞭解GI 在植物體內扮演的多重功能,實驗室先前從銀脈鳳尾蕨 (Pteris cretica cv. Albolineata) 和鐵線蕨 (Adiantum capillus-veneris L) 選殖到GI 之同源基因,分別命名為PcGI及AcGI。研究發現,由蕨類植物所選殖到的GI 同源基因同樣受生理時鐘之調控,並於光照8-12小時後表現量達最高,黎明時表現量最低。於阿拉伯芥gi突變株中大量表現蕨類之GI 基因,可以部分挽救gi突變株之晚開花性狀,亦會活化下游CONSTANS (CO)、FLOWERING LOCUS T (FT) 及SUPPRESSOR OF OVEREXPRESSION OF CONSTANS (SOC1) 之表現。為了研究GI的N端與C端在不同物種間之功能差異,將阿拉伯芥GI 之N端或C端與蕨類GI進行置換成為重組基因 (PcGI之N端加上AtGI之C端:PcAt-GI、AtGI之N端加上PcGI之C端:AtPc-GI),並將重組基因大量異位表現於gi-1突變株中,結果顯示PcAt-GI開花時間較AtPc-GI早。除此之外,進一步探討GI基因N端與C端之功能差異,分別將阿拉伯芥GI (AtGI) 和蕨類GI 同源基因 (PcGI、AcGI) 之全長及N端與C端分別大量表現於gi-1及gi-2突變株中。結果顯示,大量表現阿拉伯芥與蕨類GI之C 端於gi-1及gi-2突變株中皆有提早開花之性狀;相反地,大量表現阿拉伯芥與蕨類N端於gi-1及gi-2突變株中則只能些微提早開花時間。綜合上述結果證實,GI之C端相較於N端對植物開花扮演重要的角色。除了在開花時間上之差異外,發現在35S::AtGI和35S::AtPc-GI轉殖株中出現不稔實之性狀,由結果顯示35S::AtGI及35S::AtPc-GI可能藉由抑制數個調控花藥內壁次級細胞壁生成基因表現,如NAC SECONDARY WALL-PROMOTING FACTOR1 (NST1)、NST2及MYB DOMAIN 85 (MYB85) 而造成花藥內壁中之次級細胞壁無法形成,進而導致花藥不開裂使花粉無法釋出而無法授粉的現象,由於這兩種轉基因植物皆含有阿拉伯芥AtGI-N端片段,因此初步推測GI的N端可能具有間接影響花藥後期發育之功能而造成不稔實之性狀。
GIGANTEA (GI) has been considered to be involved in circadian clock pathway to regulate the flowering in plants. Mutation in GI delays the flowering time and increases the tolerance to oxidative stress in the Arabidopsis thaliana. To further investigate the multiple functions for GI, GI orthologues (PcGI and AcGI) were cloned from non-flowering ferns Adiantum capillus-veneris L. and Pteris cretica cv. Albolineata. The expression of fern’s GIs also showed a circadian rhythm with the highest expression at 8 to 12 h of the light period and the lowest expression at dawn. A partial rescue of late flowering phenotype by activating CONSTANS (CO), FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS (SOC1) was observed in the gi mutant of Arabidopsis ectopically expressed fern’s GIs. This implied the function of GI genes is conserved in flowering and non-flowering plants. Ectopic expression of PcAt-GI which contains the N-terminal of PcGI and C-terminal of AtGI could rescue the late-flowering of gi mutants stronger than AtPc-GI which contains the N-terminal of AtGI and C-terminal of PcGI. Furthermore, ectopic expression of C-terminal portion of GI, PcGI or AcGI promotes flowering in the Arabidopsis gi-1 and gi-2 mutants. By contrast, the N-terminal portion of these three GI orthologues could only weakly promote flowering in gi-1 and gi-2 mutants. These results revealed that the C-terminus of the GI protein is functionally more important in flowering than the N-terminus. Besides flowering time, additional male sterility of the flower due to anther indehiscence was only observed in 35S::AtGI and 35S::AtPc-GI plants. Futher analysis indicated that secondary thickening was absent in the endothecium of anther in 35S::AtGI and 35S::AtPc-GI transgenic Arabidopsis due to the down-regulation of NST1, NST2 and MYB85. Our result implied that the N-terminus of the AtGI contained the function in regulating the anther development once ectopic expressing in Arabidopsis.
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