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研究生:王秉鈞
研究生(外文):Bing-Jyun Wang
論文名稱:鐵炮百合花藥專一基因編譯一嶄新的肌動蛋白絲結合蛋白質
論文名稱(外文):The anther-specific gene encodes a novel actin-binding protein in Lilium longiflorum
指導教授:王國祥
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:2009
畢業學年度:97
語文別:中文
論文頁數:62
中文關鍵詞:百合花藥中間絲結合蛋白
外文關鍵詞:LilyantherIFABP
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LLP13是經由抑制扣除法雜合法(suppression-subtractive hybridization)從鐵砲百合花藥乾燥時期的 cDNA 集合庫中挑出的。LLP13基因全長2799 bp,可編譯區為2424 bp,可轉譯807個胺基酸。LLP13蛋白分子量為91 kDa,N端具有疏水性訊息胜肽 (signal peptide)。經序列比對分析後得知此蛋白與阿拉伯芥和水稻的中間絲(intermediate filament) 結合蛋白分別有15%和32%的相同度。選擇不含訊息胜肽的2段區域,LLP13N及LLP13C,分別含有313和337個胺基酸的片段,利用大腸桿菌大量表現,純化好的蛋白在兔體內分別產生LLP13N及LLP13C 的抗血清。LLP13C 抗血清的效價約為LLP13N抗血清的十倍。西方墨漬法分析顯示,LLP13C 及 LLP13N 抗血清都可辨認到一個大於97 kDa的蛋白質。經轉漬親合純化後的LLP13C抗體分析,發現LLP13蛋白為花藥專一,但不是熱穩定蛋白,且隨著花粉成熟逐漸累積,在成熟花粉中累積到最大量。利用二維電泳分離蛋白質與西方墨漬分析,得知LLP13蛋白質具有多型性。利用ConA-HRP(concanavalin A-peroxidase)分析得知LLP13很可能為醣蛋白。花粉萌發24小時後尚可偵測到LLP13,顯示出LLP13可能和花粉管的生長延伸有關。利用基因槍的方式將不同的Zm13構築載體送入花粉中顯示,GFP-LLP13 、LLP13-GFP及LLP13皆會影響花粉管延伸且發生嚴重扭曲的現象。含有GFP-LLP13 和LLP13-GFP的融合蛋白散布在整個花粉管中,但主要集中於花粉管前端及管中一些絲狀結構上。利用雷射共軛焦掃描顯微鏡觀察LLP13融合蛋白和mTalin-RFP在花粉管中的分布,發現有共定位(co-localization)的現象,因此推測 LLP13可能為一嶄新的肌動蛋白絲 (actin filament) 結合蛋白。已得到LLP13啟動子的部分序列,此序列含有TATA-box以及MeJA (CGTCA) 的調控序列。
The LLP-13 clone was identified from a suppression subtractive cDNA library that constructed from mRNA isolated at the desiccation stage of lily (Lilium longiflorum) anthers. The full length of LLP13 is 2799 bp in which an open reading frame of 2424 bp encodes 807 amino acid residues. The protein has a calculated molecular mass of 91 kDa and contains a hydrophobic signal peptide at the N-terminus. The protein shares 15% and 32% identities with intermediate filament binding proteins found in Arabidopsis and rice, respectively.Two pieces of fragments, LLP13N without containing the signal peptide and LLP13C (313 and 337 amino acids, respectively) were chosen and overexpressed in E. coli. The purified proteins were injected into rabbits. The titer of LLP13C antiserum is ten-fold higher than that of LLP13N. An immunoblot of total protein indicated that a protein with molecular mass larger than 97 kDa was detected either by LLP13N or by LLP13C antiserum. Antibody affinity-purified from LLP13C antiserum was used for further characterization. The LLP13 was detected as ananther-specific protein and the protein was not heat-stable .The LLP13 protein is a heterogenous glycoprotein using concanavalin A as a binding reagent.The LLP13 protein remained similar level of accumulation even after 24-h germination suggesting that the protein may play a critical role in tube growth. Particle bombardment of GFP-LLP13, LLP13-GFP and LLP13 only showed that the LLP13 exhibited protein serious bending pollen tubes. The LLP13 was preferably located both on the tip and on the cytoskeleton of pollen tubes. Cooverexpression of both LLP13-GFP or GFP-LLP13 and mTalin-RFP in pollen tube showed that the LLP13 protein and talin, an actin-binding protein were colocalized at filament of pollen tubes. It suggested that the LLP13 protein is likely a novel actin-
binding protein. The promoter region of LLP13 is 749 bp that contains TATA-box and MeJA (CGTCA) regulating sequences.
壹、中文摘要...............................................................................1
貳、英文摘要...............................................................................2
參、前人研究...............................................................................3
一、百合與花藥構造................................................................3
二、花藥/花粉發育.................................................................4
三、花粉萌發及花粉管延伸....................................................5
四、花粉管內之細胞骨架........................................................6
五、花粉專一性晚期基因........................................................7
六、花粉管細胞骨架結合蛋白.................................................9
肆、材料與方法.........................................................................13
一、材料.............................................................................13
二、表現載體的構築............................................................13
三、LLP13融合蛋白的大量表現..........................................16
四、LLP13抗血清製備........................................................17
五、西方墨點法..........................................................................17
六、百合LLP13啟動子檢測.................................................22
七、百合花粉暫時性分析....................................................24
伍、結果.....................................................................................27
一、LLP13 cDNA之特性分析..............................................27
二、LLP13抗血清製備及專一性測試...................................27
三、LLP13蛋白質之特性分析..............................................28
四、LLP13蛋白質之功能分析..............................................30
五、LLP13基因啟動子之鑑定..............................................31
陸、討論.....................................................................................32
柒、參考文獻.............................................................................35
捌、圖表.....................................................................................43
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