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研究生:宮力仁
研究生(外文):Lih-Ren Kong
論文名稱:植物中調控細胞分裂與分化的基因之選殖與功能性分析
論文名稱(外文):Molecular cloning and characterization of genes in regulating cell division and differentiation from various plant species
指導教授:楊長賢楊長賢引用關係
學位類別:博士
校院名稱:國立中興大學
系所名稱:生物科技學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:151
中文關鍵詞:阿拉伯芥晚開花突變葉片發育轉譯調控
外文關鍵詞:late-floweringfld-2Zinc fingerEAR domainAtEPF1p70s6k40S ribosomal protein S6
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fld-2為一個由EMS突變劑誘變而來的晚開花突變。FLD基因位於第三條染色體上端約13cM的位置,介於g5970到Athch1b之間。此區域含有F13M14、T7M13、F9F8、F11B9 等四個BAC clones。透過(1) subcloned這四個BAC clones來互補fld-2突變株,並觀察轉殖株之開花時間,(2)觀察位於g5970到Athch1b之間的T-DNA tagged lines的開花時間有無延後,(3)以proteomic approach比較在野生型阿拉伯芥與fld-2晚開花突變種之間有無差異,(4)比較特定基因之序列在野生型阿拉伯芥與fld-2晚開花突變種中有無差異等四種方式來選殖FLD基因。FLD基因的確實位置是F13M14.34(At3g10390),其含有兩個exons,可轉譯出含789個氨基酸之蛋白質。FLD在靠近N端的位置含有屬於SWIRN(SWI3p、Rsc8p以及Moira)的domain,與protein-protein interaction有關。FLD含有屬於polyamine oxidase的domain,與chromatin的deacetylation有關。fld-2突變體的突變點是在2149 bp的位置由C變成T,造成Glutamic acid變成stop coden而提早終止轉譯。FLD之mRNA在不同天數之植株以及不同器官中均有表現,僅有表現量高低的差異。其在花軸與根部表現量最低,在營養葉與托葉表現量中等,而在16及24天大之全株植物樣本以及花苞中有最高的表現量。本研究並進行其他植物之FLD同源基因選殖,結果選殖出文心蘭的OnFLD、蝴蝶蘭的PeFLD以及青花菜的BoFLD。(第二章)
本實驗另進行阿拉伯芥AtEPF1基因之選殖與分析。AtEPF1為Cys2/His2型的Zinc finger基因,其蛋白質與矮牽牛中的EPF1有34%的相同性與45%的相似性。AtEPF1具有專屬於EPF family的完整的保守序列CX2CX3FX5LX2HX3H,以及屬於Cys2/His2 ZFP的核心序列QALGGH。除了zinc-finger motifs,AtEPF1的C端中還帶有類似於EAR (ERF-associated amphiphilic repression)的domain。AtEPF1在花苞中表現量最高,在24天大的seedling中表現量較低,在8天、16天大的seedling以及rosette leave中則表現量極低。在不同大小的花苞中,AtEPF1只表現在大花苞及全開的花,在小花苞及中花苞則無任何表現量。35S::AtEPF1之轉基因阿拉伯芥,在外觀上可觀察到葉片異常突出且捲曲及植株矮化的現象。透過觀察AtEPF1在C端外加mGFP後於細胞內所在的位置,發現AtEPF1是集中於細胞核的位置,因此可以肯定AtEPF1具有進入細胞核的能力。進一步分析發現,AtEPF1可能是透過對BOP2基因進行負調控,進而影響到分生組織的生長,隨後再影響到葉片的發育。(第三章)
p70 ribosomal S6 kinase (p70s6k) 透過phosphorylated S6 蛋白質進而調控5''TOP mRNAs 之轉譯及細胞周期之進行。本研究對阿拉伯芥中的p70S6K (ATPK6與ATPK19)及40S ribosomal protein S6 (AtS61與AtS62)基因進行選殖及分析。ATPK6的mRNA主要表現在16天大之全株植物、托葉及花苞,在營養葉及花軸只有微弱的表現量。ATPK19的mRNA主要表現在托葉及花軸,在營養葉及花苞只有微弱的表現量。AtS61的mRNA在各個部位均有表現,只是在營養葉及花軸的表現量較弱。AtS62的mRNA在各個部位均有表現,且彼此間表現量的差異不大。在轉殖35S::ATPK19、35S::AtS61或35S::AtS62之阿拉伯芥中,可觀察到花瓣與雄蕊在發育時因無法正常伸長,結果造成受孕困難,果莢發育不完全。在轉殖35S::AtS61 antisense之阿拉伯芥中,觀察到外觀較野生種阿拉伯芥來的矮小,嚴重者甚至小至十倍以上。透過分析受到IAA刺激的阿拉伯芥花苞中的5''TOP mRNA表現量,發現AP3、PI應該是直接受到p70S6K與S6之調控,而NAP是因為AP3、PI表現量的提昇而間接受到影響。利用PCR的方式來測試AP3與PI的轉錄起始點,發現AP3與PI的轉錄起始點應該是位於5’-UTR中的polypyrimidine sequence的外側。(第四章)
fld-2 mutant, significantly delayed flowering, was isolated from Arabidopsis. FLD was mapped on top arm of chromosome 3 between molecular markers g5970 and Athchlb. Four BAC clones in this region were subcloned for further complementation test. T-DNA tagged lines for genes in this region were obtained and phenotype analyzed. A proteomic approach was also applied to analyze the protein expression profiling in wild-type plants and fld-2 mutant. FLD is locus At3g10390 that consists of two exons and encodes a protein of 789 amino acid residues. FLD contain a region referred to as a SWI3p, Rsc8p, and Moira (SWIRM) domain in the N-terminal region that involved in protein-protein interaction. FLD also contain a polyamine oxidase domain that involved in chromatin deacetylation. A point mutation (C to T) that converts Glu716 (CAG) into a premature stop codon (TAG) was found in fld-2 mutant. FLD was expressed in all organs and tissue tested. Homologues of FLD were cloned from Oncidium Gower Ramsey、Phalaenopsis amabilis var. formosana and Brassica oleracea var. italica. (Chapter 2)
AtEPF1, showed high homology to the EPF family gene, was isolated and characterized from Arabidopsis. AtEPF1 showed high sequence identity to Petunia EPF1. A conserved sequence containing a core QALGGH motif identified in the Cys2/His2 zinc finger region of the AtEPF1 was present in EPF family genes. Besides, AtEPF1 contain of an amphiphilic amino-acid sequence similar to the EAR motif in its C-terminal region. AtEPF1 mRNA was detected in floral buds as well as in 24day-old seedling. Interestingly, in floral buds, AtEPF1 only expressed in mature floral buds and open flowers. Ectopic expression of AtEPF1 in transgenic Arabidopsis plants showed novel phenotypes by producing ectopic outgrew and curled rosette leaves. Further analysis indicated that AtEPF1 proteins were able to enter nucleus. These results indicated that AtEPF1 may play a role in regulating meristematic activity in the embryo development or early stage of the leaf development by modulating expression of BOP2 gene. (Chapter 3)
S6 protein phosphorylated by p70 ribosomal S6 kinase (p70s6k) signaling pathway plays a key role in regulating cell cycle by translationally regulating specific 5''TOP mRNAs. In this study, p70S6K (ATPK6、ATPK19) and 40S ribosomal protein S6 (AtS61、AtS62) genes were cloned and characterized from Arabidopsis. Ectopically expressed ATPK19, AtS61 and AtS62 genes caused abnormal flower development with short petals and stamens in transgenic Arabidopsis plants. Ectopically expressing antisense of AtS61 caused a severe reduction (more than 10 times) in plant size in transgenic plants. Further sequence analysis was used to identify putative 5''TOP mRNAs regulated by p70S6K signaling pathway especially for genes involving in petal and stamen development such as AP3 and PI. (Chapter 4)
第一章 緒言

- 植物基因體學之發展現況 1
- 顯花植物的生活史 2
- 植物的開花誘導途徑 2
- 植物的葉片發育 3
- 植物的花朵發育 4
- 參考文獻 6
- 圖表 15

第二章 開花時間相關基因FLD之染色體定位與分子選殖

- 摘要 17
- 前言 18
- 材料與方法 24
- 結果 36
- 討論 43
- 參考文獻 49
- 圖表 57

第三章 阿拉伯芥中參與葉型發育之EPF基因之選殖與分析

- 摘要 82
- 前言 83
- 材料與方法 86
- 結果 90
- 討論 94
- 參考文獻 97
- 圖表 103

第四章 阿拉伯芥中p70核醣體S6激酶以及40S核醣體蛋白質S6參與調控花器形成之研究

- 摘要 112
- 前言 113
- 材料與方法 116
- 結果 119
- 討論 124
- 參考文獻 128
- 圖表 135

第五章 結論與展望

- 參考文獻 151
第一章 緒言

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第二章 開花時間相關基因FLD之染色體定位與分子選殖

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第三章 阿拉伯芥中參與葉型發育之EPF基因之選殖與分析

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第四章 阿拉伯芥中p70核醣體S6激酶以及40S核醣體蛋白質S6參與調控花器形成之研究

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第五章 結論與展望

周明倫. 2000. 擬南芥中調控發育時期轉換相關基因之遺傳探討及分子選殖. 國立中興大學農業生物科技學研究所博士論文.

Blázquez. M. A. (2000). Flower development pathway. Cell Sci. 3547-3548.

Elda B. P., Raul A., Adriana O. L., Tzvetanka D. D. and Jimenez. E. S. (2002). Auxin stimulates S6 ribosomal protein phosphorylation in maize thereby affecting protein synthesis regulation. Physiol. Plant. 115:291-297.

He, Y., Michael, S. D. and Amasino, R. M. (2003). Regulation of flowering time by histone acetylation in Arabidopsis. Science 302, 1751-1754.
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