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研究生:謝秉翰
研究生(外文):Ping-Han Hsieh
論文名稱:一個具有Agenet和ENT domain的蛋白質參與調控水稻種子萌芽及產量
論文名稱(外文):Identification of a Novel Agenet and ENT Domain-Containing Protein Involved in Seed Germination and Grain Yield in Rice
指導教授:余淑美余淑美引用關係
指導教授(外文):Su-May Yu
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生命科學系暨基因體科學研究所
學門:生命科學學門
學類:生物訊息學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:中文
論文頁數:71
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水稻不僅是全世界一半以上人口的主食,也是唯一在淹水環境下還能生長的作物。藉由了解水稻耐淹水的機制能夠改善並增進其他作
物的產量。
SnRK1A能夠偵測植物體內的能量代謝,並調控缺糖訊息傳遞路徑以及無氧發酵作用的進行,而我們實驗室在先前的研究發現了CIPK15這個重要的基因位於SnRK1A的上游,在水稻耐缺氧中扮演重要的角色。為了要更加了解這條路徑如何調節,藉由forward genetics approach的方式來篩選出跟淹水有關的T-DNA插入水稻突變株。我們發現一個具耐淹水性狀的突變株M30505,經過資料庫比對T-DNA插在Os03g58190這個基因開始轉譯的密碼子上游1384 bp的位子,這個基因被T-DNA上35S啟動子所活化。同時我們也觀察到M30505在田間生長有產量變多,百粒重增加的性狀產生。M30505也表現出在淹水環境下以及黑暗中生長較佳的現象。我們也發現CIPK15-SnRK1A signaling pathway在M30505中也會被活化,這代表Os03g58190可能位於這條路徑的上游。
Os03g58190大量表現於開花組織,含有2個非常保守的domain:Agenet domain和Emsy N Terminus(ENT)domain,而這2個domain的功能目前在植物中仍然未知。然而在無氧的環境下Os03g58190的表現量會減少,但能藉由外加糖恢復;另外在萌芽期間Os03g58190在胚中會從乾胚時的高表現在發芽後減少表現量。這些結果顯示Os03g58190受缺氧和缺糖所抑制。為了更加確定Os03g58190的功能,我們可以藉由過量表現與干擾表現Os03g58190的轉殖株來幫助我們了解。

Rice (Oryza Sativa) feeds approximately 50% of the world’s population, and is also the only major crop that can germinate under flooding conditions. Understanding the mechanism of flooding tolerance in rice would facilitate the improvement of different
varieties of rice as well as other crops.

Previous study in our lab indicated that CIPK15 acts upstream of the global energy sensor SnRK1A-dependent sugar starvation signaling and fermentation pathways, and plays a key role in the O2 deficiency tolerance in rice. To further elucidate the detailed mechanisms regulating these pathways, a forward genetics approach was employed by screening T-DNA activation/knockout tagged rice mutants. One flooding tolerance mutant, M30505, which carries a T-DNA insertion at position 1384 bp upstream of the translation start codon of an expressed gene, Os03g58190, with unknown function was identified. Expression of this gene is activated by the 35S promoter in the T-DNA. M30505 homozygote has more grain yield and 100-grain weight than segregated wild-type grow in paddy. M30505 also exhibits a phenotype of enhanced seedling growth under flooding and dark conditions. The CIPK15-SnRK1A signaling pathway is also activated in M30505, indicating that Os03g58190 acts upstream of these genes.

Os03g58190 is highly expressed in floral tissue. This gene encodes a protein with two conserved domains, the Agenet and Emsy N Terminus (ENT) domains. The function of these two domains in plant is unknown. Under flooding conditions, the expression of Os03g58190 is reduced in embryos but could be recovered by sugar. In air, the expression of Os03g58190 is high in dry embryos but decreased after seed imbibition. These studies indicate that the expression of Os03g58190 is suppressed by O2 deficiency and sugar starvation. To confirm the function of Os03g58190, gain- and loss- of function approach by generating Os03g58190 overexpression and RNAi transgenic plants is currently conducted.

目 錄
致謝…………………………………………………….…………….…...i
中文摘要………………………………….……………………………...ii
英文摘要……………………………………..………………….............iv
目錄……………………..……………………………………………......v
圖表目錄………………..………………………………………..…..….vi
壹、前言…………………………………………………………..………1
貳、前人研究………………………………………………………..……2
叁、材料與方法……………………………………………..……………9
肆、結果…………………………………………………………………21
伍、討論………………………………………………...……………….30
陸、參考文獻……………………………………………………………66
附錄…………………………………………………………………..…69


圖 表 目 錄

Figure 1. Screening process for identification of M30505………….……….34

Figure 2. Genotyping identified T-DNA insertion M30505 homozygote (-/-) and heterozygote (+/-)………….………………….…………..36

Figure 3. Grain yield of M30505 homozygote is increased……………………….38

Figure 4. Growth of M30505 is more tolerant to flooding.…………...……..........40

Figure 5. An expressed protein is activated in M30505..…………………………42

Figure 6. The expressed protein activates the CIPK15-SnRK1A signaling pathway..…………………………………………………………………………….48

Figure 7. The expressed protein promotes seedling growth..……….……………52

Figure 8. The expressed protein contains two conserved domains..……………..54

Figure 9. The expressed protein is highly expressed in flowering tissue. ……….56

Figure 10. The expressed protein is relocated from nucleus to the cytoplasm under sugar starvation. ………………………………………………….…………58

Figure 11. Overexpression of the expressed protein activats the αAmy3 promoter..................................................60

Figure 12. The expression of expressed protein is repressed by anoxia and recovered by sugar.……………………………………………………..………...…62

Table 1. Primer list…………………………….......................................64



參考文獻

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Hernandez, J.M., Feller, A., Morohashi, K., Frame, K., and Grotewold, E. (2007). The basic helix loop helix domain of maize R links transcriptional regulation and histone modifications by recruitment of an EMSY-related factor. Proc Natl Acad Sci U S A 104, 17222-17227.
Hiei, Y., Ohta, S., Komari, T., and Kumashiro, T. (1994). Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. The Plant journal : for cell and molecular biology 6, 271-282.
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