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研究生:洪士賢
研究生(外文):Shin-Hsien Hung
論文名稱:阿拉伯芥Myo-Inositol-1-PhosphateSynthase基因之特性分析
論文名稱(外文):Characterization of Arabidopsis Myo-Inositol-1-Phosphate Synthase gene
指導教授:吳文鑾
指導教授(外文):Wen-Luan Wu
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物學系碩博士班
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2003
畢業學年度:91
語文別:中文
論文頁數:59
中文關鍵詞:阿拉伯芥胚胎發育基因
外文關鍵詞:Arabidopsis thalianaembryogenesisgene
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MIPS (myo-inositol-1-phosphate synthase)酵素於生物體內扮演重要的生理角色,如負責催化D-glucose 6-phosphate,使其轉化為MI-1-P (myo-inositol-1-phosphate),進一步參與許多生長發育反應。本實驗即以MIPS基因為研究對象,進行阿拉伯芥AtMIPS1 (Arabidopsis thaliana myo-inositol-1-phosphate synthase 1)基因之遺傳分析,探討其在胚發育過程所扮演角色。首先經由Nottingham Arabidopsis stock center (NASC)取得以T-DNA insertion方式所形成的mips1突變株。利用掃描式電子顯微鏡及光學顯微鏡觀察,發現此突變株種子,其種皮上之細胞呈現不規則排列的情形,且突變種子中亦觀察不到成熟胚之存在;證實此突變性狀的發生,確與T-DNA插入AtMIPS1基因有關。此外向Salk Institute Genomic Analysis Laboratory (SIGnAL)取得另一Salk序號之mips1突變之種子進行培養,觀察後發現其與本研究對象mips1突變株具有相同性狀,因此證明突變種子的產生,確實是AtMIPS1基因突變所造成之結果。利用南方墨點法分析阿拉伯芥MIPS1基因拷貝數目,以AtMIPS1基因譯碼區為探針,在多個限制酵素作用下,僅出現1個雜合反應條帶,因此推測AtMIPS1基因為單一拷貝基因。
為確認突變型種子為何種基因型?培養mips1突變株T2植株,收取並種植正常型種子後,待其長成外表型亦正常之T3植株,收取果莢內之種子,並觀察其種子外表型。發現果莢內正常型與突變型種子的比例皆接近3:1 (X2=3.841 , P < 0.05)。符合孟德爾之遺傳分離率,證實果莢內正常型的種子應由MIPS1/MIPS1與MIPS1/mips1所組成,而突變型種子則為mips1/mips1。
進一步讓mips1突變株T2植株進行自交,將具有突變種子的植株其花粉作為父本,野生型阿拉伯芥作為母本,雜交後種植果莢內之種子,結果發現有突變種子的出現,因此證明mips1可經由花粉傳遞,故為核內遺傳。而利用光學顯微鏡觀察以透明法處理之突變胚,發現發生變異的時期為球形胚發育至心形胚之階段,而突變心形胚會發生無胚柄構造的現象,推測胚發育延遲及變形的原因為胚分裂時細胞分裂的方向錯誤,而造成無胚柄構造的結果,而以GUS染色分析基因表現位置,AtMIPS1基因表現主要是在胚,這些結果顯示AtMIPS1基因在阿拉伯芥胚胎發育過程中的確扮演重要之功能。
The MIPS (myo-inositol-1-phosphate synthase)enzyme plays an important physiological role in organisms, catalyzing D-glucose 6-phosphate to MI-1-P (myo-inositol-1-phosphate) that participates many development processes. The genetic analysis of AtMIPS1 gene of Arabidopsis thaliana and examination of its function in embryogenesis were perfomed. The seeds of mips1 mutant, generated by T-DNA were provided by NASC. Using scanning electron microscopy and light microscopy to observe seed morphology, the cell arrangement of seed coat was abnormal and failed to develop to mature embryo. Another mips1 mutant had the different Salk number which obtained from SIGnAL with the same phenotype, proved that the mutant seeds were also caused by the mutation of AtMIPS1 gene. Southern blotting of genomic DNA with AtMIPS1 coding region suggested that AtMIPS1 was a single copy gene in Arabidopsis thaliana.
To determine the genotype of mutant seeds, seeds from siliques of normal phenotype T3 plant that cultivated from the normal seeds of T2 plants which had mips1 mutant were collected. The ratio of normal seeds to mutant seeds in each silique were close to 3:1 (X2 = 3.841, P < 0.05), fitting to the law of segregation. This result confirmed that the genotype of normal and mutant seeds in siliques were MIPS1/MIPS1(or MIPS1/mips1) and mips1/mips1, respectively.
T2 plant were further allowed to self-pollinate, pollens from plant which had mutant seeds cross to wild type serving as maternal parents. The presence of mutant seeds in siliques from hybriding plants indicated that mips1 was transmitted by pollens. In development processes, the embryo of mutant seed which occurred variants during the stage of globular to heart embryo was observed by using light microscopy. The phenomenon of non-suspensor in mutant heart embryo may be caused by the wrong direction of cell division in embryogenesis. The AtMIPS1 promoter::GUS expression was detected only in embryo. These results suggest that AtMIPS1 gene may play a critical role in embryo development of Arabidopsis thaliana.
中文摘要....................................................i
英文摘要...................................................ii
誌謝......................................................iii
目錄.......................................................iv
表目錄...................................................viii
圖目錄.....................................................ix
縮寫名詞對照表..............................................x
第一章 緒論................................................1
一、Myo-Inositol-1-phosphate synthase (MIPS1)之簡介.........1
(一) MIPS基因結構........................................1
(二) MIPS基因之研究簡史..................................1
(三) MIPS基因之生理功能..................................2
(四) 高等植物的MIPS基因相關研究..........................2
(五) 離層酸與MIPS基因表現之關係..........................3
二、胚發育簡介..............................................4
(一) 高等植物之有性生殖方式..............................4
(二) 胚發育過程..........................................4
(三) 阿拉伯芥的胚發育....................................5
三、阿拉伯芥胚發育基因的相關研究............................6
四、研究之源起與目的........................................6
第二章 材料與方法..........................................8
一、實驗材料................................................8
二、實驗方法................................................8
(一) mips1之遺傳分析.....................................8
1. 種植阿拉伯芥..........................................8
2. 遺傳分離率分析........................................8
3. 遺傳特性分析..........................................9
(二) 阿拉伯芥野生型植株及突變植株mips1種子外觀之觀察.....9
1. 解剖顯微鏡觀察正常與突變種子..........................9
2. 掃描式電子顯微鏡檢 (SEM)..............................9
(三) 阿拉伯芥野生型植株及突變植株mips1胚發育之觀察......10
1. 石蠟切片法...........................................10
2. 透明法...............................................10
(1) 胚生長時期的區別.................................10
(2) 固定.............................................10
(3) 觀測發育中胚之內部型態...........................11
(四) 阿拉伯芥基因組DNA的萃取............................11
1. 大量萃取阿拉伯芥基因組DNA......................11
2. 微量萃取阿拉伯芥基因組DNA......................12
(五) 南方氏雜合實驗 (Southern hybridization)............12
1. 隨機引子探針的製備.............................12
2. 阿拉伯芥基因組DNA的限制酵素切割作用............13
3. 南方轉漬實驗...................................13
4. 雜合反應.......................................14
(六) 微量製備質體DNA...................................14
1. 培養細菌.......................................14
2. 微量抽取.......................................14
3. 限制酵素作用測試...............................15
4. 由瓊脂凝膠中回收DNA片段........................15
(七) 聚合酵素連鎖反應─擴增阿拉伯芥MIPS1 promoter......15
1. 聚合酵素連鎖反應...............................15
(八) 轉殖阿拉伯芥植株..................................16
1. 聚合酵素連鎖反應擴增阿拉伯芥AtMIPS1 promoter...16
2. 構築yT&A- AtMIPS1 promoter質體.................16
3. 大腸桿菌DH5a菌株熱休克(heat shock)轉型.........16
4. 微量抽取yT&A- AtMIPS1 promoter質體DNA..........17
5. 構築pBI121-AtMIPS1 promoter質體................17
6. 大腸桿菌DH10B菌株熱休克轉型....................17
7. 微量抽取pBI121-AtMIPS1 promoter質體DNA.........18
8. 農桿菌 (Agrobacterium tumefaciens) LBA4404菌株
電穿孔轉型.....................................18
9. 阿拉伯芥轉基因啟動子實驗.......................18
(九) GUS染色...........................................19
第三章 結果...............................................21
一、阿拉伯芥mips1突變株性狀之觀察..........................21
二、阿拉伯芥AtMIPS1基因遺傳分析............................22
(一) 遺傳分離率分析....................................22
(二) 基因型分析........................................23
(三) 遺傳特性分析......................................24
三、阿拉伯芥mips1突變株之胚胎發育情形......................24
四、阿拉伯芥AtMIPS1基因組織特異性表現分析..................25
第四章 討論...............................................27
一、阿拉伯芥mips1突變株之性狀特性..........................27
二、阿拉伯芥AtMIPS1基因族系組成與遺傳分析..................28
三、阿拉伯芥mips1突變株之胚胎發育探討......................29
四、阿拉伯芥AtMIPS1基因之表現分析..........................30
第五章 未來展望...........................................31
第六章 參考文獻...........................................32
附錄.......................................................58
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