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研究生:吳佳娟
研究生(外文):Wu, Chiachuan
論文名稱:運用大量表現與雙重突變之策略探討阿拉伯芥BPM 基因的功能
論文名稱(外文):Functional analysis of Arabidopsis BPM genes by over-expression and double mutation
指導教授:黃鐘慶黃鐘慶引用關係
指導教授(外文):Huang, Jongchin
口試委員:樊琳洪志勳
口試委員(外文):Farh, LynnHung, Chihhsin
口試日期:2013-01-14
學位類別:碩士
校院名稱:國立屏東教育大學
系所名稱:化學生物系碩士班
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:42
中文關鍵詞:花粉雄配子體蛋白質降解雙重突變
外文關鍵詞:Pollenmale gametophyteubiquitin-proteasome systemdouble mutation
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有性生殖是藉由精卵結合而得以完成,在開花植物中,花粉會藉由不同方式散播並附著於柱頭上,此時花粉管便會開始萌發生長以啟動授粉的程序,故開花植物的繁衍乃是藉由花粉-也就是雄配子體(male gametophyte)的傳播而進行。花粉在整個授粉過程中有著產生精細胞及將精細胞傳送至胚囊以完成雙重授精(double fertilization)之重要任務,所以花粉的發育對於開花植物的有性生殖是很重要的。細胞內蛋白質降解具有不同之機制,近年發現某些含有BTB/POZ 功能區之蛋白質會與跟CUL3 產生交互作用,以單一蛋白質形成另一種不同SCF(Skp1–Cul1–F-box) 以及ECS(ElonginC–Cul2–SOCS box) complex 形式的cullin-based ubiquitin ligase,進而參與Ub/26S proteasome分解蛋白質之途徑以調控發育過程。在實驗室前人針對AtBPM2基因的突變株(SALK_121270)鑑定分析實驗中,發現阿拉伯芥AtBPM2單一基因突變並未對阿拉伯芥表現型產生顯著影響,突變株仍然可以正常繁衍後代,意味著可能有其它基因互補AtBPM2基因的功能。由於AtBPM2 與AtBPM3在花粉的精細胞有相對高的表現量,因此本研究想藉傳統遺傳學方式,用人工授粉與自花授粉方式,使植株同時帶有AtBPM2 與AtBPM3兩基因突變,以雙重突變的方式觀察當AtBPM2 與AtBPM3兩基因皆突變後是否會對花粉發育或功能產生影響。同時,再以大量表現之方式將AtBPM2基因於其他部位表現,觀察是否有任何表現型的改變,來推測其可能的功能。結果發現,大量表現AtBPM2基因,導致早開花之現象出現,而雙重突變的結果尚須進一步鑑定。
Pollen grain is the male gametophyte of flowing plant and is necessary for successful double fertilization during plant sexual reproduction. Therefore, pollen development is very critical in plant. A cDNA (LLBTB) cloned from lily pollen shows high identity to the Arabidopsis gene family (AtBPM1~6) whose function is unclear. Both deduced amino acid sequences contain BTB/POZ domain which plays important role in protein-protein interaction. AtBPM2 and AtBPM3 express highly in sperm cells. It has been suggested that proteins containing BTB/POZ domain will be involved in protein degradation mechanism of 26S proteasome by interacting with CUL3 protein to form an E3 ubiquitin ligase. Specific protein substrate will be ubiquitined and degraded in 26S proteasome. To investigate the possible function of AtBPM2 and AtBPM3 in sperm cells, overexpression and double mutation strategies are taken to analyze both genes’ function. AtBPM2 overexpression plants show early flowering phenotype, and the result of double mutation (AtBPM2 and AtBPM3) is not confirmed yet.
摘要 3
文獻探討 5
開花植物的生殖與花粉的發育 5
泛素蛋白質降解系統與BTB/POZ domain 蛋白質之關聯 6
材料與方法 11
阿拉伯芥之種植條件與材料收集 11
質體DNA的萃取 11
聚合酶鏈鎖反應(PCR) 11
TA cloning 12
DNA回收與純化 12
PCR產物的純化與回收 12
重組DNA之構築 13
勝任細胞的製作 13
轉形(transformation) 13
農桿菌勝任細胞的製作 14
農桿菌轉形 14
阿拉伯芥之基因轉殖 14
阿拉伯芥轉殖株的篩選 14
植物基因組DNA的萃取(genomic DNA extraction) 15
RNA的萃取 15
RNA於電泳膠片(denatured gel)的觀察 15
RNA反轉錄聚合酶鏈鎖反應(RT-PCR) 16
實驗結果 17
建立鑑定並分析基因大量表現之植株 17
阿拉伯芥T-DNA插入突變株鑑定與雙重突變株之建立 17
討論 19
實驗圖表 20
附錄 33
參考文獻 37

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