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研究生:楊正守
研究生(外文):Cheng-Shou Yang
論文名稱:鐵炮百合兩個嶄新的花藥專一基因與其蛋白質之特性分析
論文名稱(外文):Characterization of Two Novel Anther-specific Genes and Their Encoding Proteins in Lilium longiflorum
指導教授:王國祥
指導教授(外文):Co-Shine Wang 
學位類別:碩士
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:2008
畢業學年度:96
語文別:中文
論文頁數:49
中文關鍵詞:鐵炮百合花藥氈層花粉外壁
外文關鍵詞:Lilium longiforumanthertapetumexine
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  • 被引用被引用:1
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LLA-142和LLA-1271是利用抑制扣除雜合法(suppression subtractive hybridization)從鐵炮百合小孢子發育時期的花藥cDNA集合庫中挑出的cDNA片段。LLA-142和LLA-1271利用5′與3′-RACE得到全長的序列。LLA-142全長為379 bp,可轉譯出56個胺基酸,估計蛋白質分子量為6.2 kDa。LLA-1271 cDNA可發現兩條類似的序列,LLA-1271a全長為970 bp可轉譯出225個胺基酸,而LLA-1271b全長為 879 bp可轉譯出226個胺基酸,其差異為LLA-1271a在ORF少了3 bp,而LLA-1271b在3′-UTR缺少了一段76 bp的序列。LLA-1271扣除N端26個胺基酸訊息胜肽,估計成熟蛋白質分子量為21 kDa,整個蛋白質為八個高度保留的重複序列所涵蓋。LLA-142和LLA-1271經由北方墨漬法分析,顯示出兩基因都為小孢子時期花藥專一性的基因,在小孢子及花藥壁都有表現。進一步利用原位雜合(in situ hybridization)的實驗確定LLA-142和LLA-1271在花藥的绒氈層表現,在花藥發育的早期(花苞2 cm)並無偵測到兩基因的訊號,3-4 cm花苞時在花藥的绒氈層有明顯的訊號,5 cm花苞時绒氈層的兩基因訊號減弱,顯示出兩基因表現的強弱隨著绒氈層發育同步消長。LLA-1271 mRNAs的表現受內生和外加的激勃素誘導,而LLA-142不受外加的激勃素所調節。利用激勃素抑制劑uniconazole和乙烯抑制劑2,5-norbornadiene處理,顯示LLA-1271受激勃素和乙烯的調控,並且乙烯和激勃素相互影響LLA-1271的表現。利用西方墨漬法分析,發現LLA-142和LLA-1271為小孢子時期花藥專一性的蛋白質。利用花藥分層分析顯示在處理過0.5%或2% Triton X-100的小孢子之壁蛋白中,可偵測到LLA-1271蛋白訊號。LLA-1271累積在小孢子發生有絲分裂之前的外壁上,推測其蛋白質可能參與小孢子外壁早期的發育。利用TAIL-PCR找到LLA-142基因的啟動子序列966 bp,其中包含了TATA-box、CAAT-box、七個花粉專一調控區(AGAAA、TCAC)和荷爾蒙相關的調控區。
目錄

壹、前言 ………………………………………………………………1
貳、前人研究 …………………………………………………………2
ㄧ、花藥的構造和發育 ………………………………………………2
二、絨氈層與花粉壁的發育 …………………………………………3
三、花藥專一性基因 …………………………………………………5
四、花藥專一性基因啟動子和雄不稔 ………………………………8
五、花藥的發育受荷爾蒙的調控……………………………………11
參、材料與方法………………………………………………………13
一、材料和處理方法…………………………………………………13
二、總RNA的萃取與mRNA的純化…………………………………13
三、5′與3′-RACE-PCR…………………………………………15
四、北方墨漬法(Northern blot)………………………………18
五、原位雜合(In situ hybridization)………………………20
六、LLA-142融合蛋白的大量表現(Overexpression)…………21
七、西方墨漬法(Western blot) ………………………………22
八、百合LLA-142啟動子的鑑定…………………………………28
肆、結果………………………………………………………………32
一、LLA-142和LLA-1271基因之特性分析 …………………………32
二、LLA-142和LLA-1271蛋白質之特性分析 ………………………35
三、LLA-142基因啟動子之鑑定 ……………………………………38
伍、討論………………………………………………………………40
陸、參考文獻…………………………………………………………44
柒、圖表………………………………………………………………51
曾稚鐙。2006鐵炮百合花藥發與早期基因之選殖與特性分析。國立中興大學碩士論文。
葉芳凌。2008鐵炮百合兩绒氈層基因LLA-1271和LLA-115之特性分析和表現。國立中興大學碩士論文。
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