博碩士論文 972204023 詳細資訊




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姓名 莊鎔昀(Jung-Yun Chuang)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 利用化學遺傳法研究阿拉伯芥 revert to eto1 41 (ret41) 之功能研究
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摘要(中) 乙烯是一種化學結構簡單的氣體荷爾蒙。在高等植物中,乙烯參與調控植物的生長和發育。為了瞭解更多乙烯的相關的訊息,利用化學遺傳法的方式使用小分子干擾乙烯的反應而篩選阿拉伯芥突變株,並進一步探討突變之基因和乙烯路徑的關係。
依據乙烯三相反應的外表型,我們從10000種小分子中篩選出一群小分子,其主要的化學結構為喹唑啉酮,這些小分子化合物可有效的抑制 ethylene overproducer 1 (eto1)白化苗的三相反應。因此,利用這些小分子篩選的阿拉伯芥突變株,我們稱為revert to eto1 (ret),因為在小分子化合物作用下,ret突變株外表型可回復成像eto1。我們分別由基因功能活化庫和化學誘變之阿拉伯芥突變庫中各挑選出7個和13個ret突變株。
而我論文主要的研究對象是一個從基因功能活化庫挑選出的ret突變株,稱為ret41。ret41突變株除了可以在含有小分子化合物之培養基,表現型回復成三相反應之外,還有一些特殊的外表型,包含矮小之植株、圓形之叢生葉、降低受孕率、缺少頂芽優勢和花期較晚等性狀。經由回交之後的遺傳分析結果顯示,ret41為單一隱性突變株。我利用南方轉漬法證實ret41有5個嵌入點,接著利用TAIL-PCR和質體救援法的方式找出了兩個嵌入點。然而,基因鑑定分析的結果,這兩個嵌入點無法和ret41的外表型吻合,顯示找到的這兩個嵌入點並非真正影響ret41外表型之突變點。另外,ret41白化苗的外表型在乙烯的生合成抑制劑中仍有三相反應,顯示ret41的功能可能在乙烯受體的下游。同時,我也發現ret41的外表型不需依賴eto1-5的突變,而且ret41也不同於ctr1-1的突變。同型結合子的ret41突變株顯示出無法受孕的性狀。然而,ret41的花粉外觀和活性跟野生型和eto1相比並無顯著差異,但ret41的花藥較野生型小且花絲也較野生型短。以電子顯微鏡所進行的細部觀察,我發現柱頭不正常的發育和雄蕊跟雌蕊不同的生長時期,可能是造成ret41無法自花授粉的原因。未來的工作方向,將是尋找ret41之突變基因並了解該基因之功能,所得到的資訊,可以幫助我們了解乙烯參與在植物不同發育時期的作用。
摘要(英) Ethylene is a gaseous phytohormone with a very simple chemical structure. In higher plants, ethylene is involved in regulation of many aspects of plant growth and development. To gain more understanding about ethylene biology, a chemical genetics approach was employed to first screen for small molecules interfering with ethylene response in Arabidopsis thaliana and subsequently to identify and characterize mutants resistant to the hit compounds.
A group of small molecules with a quinazolinone backbone were identified from a phenotype-based screening of a collection of 10,000 chemical compounds to score suppression of the triple response in etiolated ethylene overproducer 1 (eto1) seedlings. These identified small molecules, named hit compounds, were used to screen Arabidopsis mutants, named revert to eto1 (ret), with restoration of the eto1 phenotype in the presence of hit compounds. There were 7 and 13 ret mutants were identified from collections of Arabidopsis seeds mutagenized by T-DNA insertional activation tagging and ethane methyl sulfonate (EMS) , respectively.
The main objective of my thesis research is to characterize one of the ret mutants, ret41, which was identified from the T-DNA activation tagging mutant collection. In addition to exhibit constitutive triple response in the presence of hit compounds, ret41 has a complex phenotype consisting of dwarf stature, round leaves, reduced male fertility, loss of apical dominance and delayed flowering time in rosette plants. Genetic analysis indicates that ret41 is a single allele recessive mutation. Results from Southern blot analysis indicate that there are 5 T-DNA insertions. By using TAIL-PCR (Thermal asymmetric interlaced PCR) and plasmid rescue methods, I identified 2 of the insertion sites. However, neither of the insertions co-segregates with the phenotype of ret41 by genotyping data, suggesting that the mapped insertions are not likely responsible for ret41 phenotype. Further analysis revealed that the triple response phenotype in etiolated ret41 seedlings was maintained in the presence of inhibitor of ethylene biosynthesis and antagonist of ethylene perception, which suggests that RET41 may function downstream of ethylene receptors. Moreover, the etiolated phenotype of ret41 is independent of eto1-5 allele and ret41 is not allelic to ctr1. Homozygous ret41 mutant showed a severe defect in fertility to produce seeds. There is no significant difference in pollen viability and morphology between ret41 and wild type or eto1 plants. However, the anthers of ret41 are smaller and the pistils are shorter than those in wild type. Microscopic analyses suggested that unusual development of stigmata and differential growth of stamens and pistils are plausible causes for unsuccessful pollination in ret41 mutant to result in sterility. Future work to clone and characterize the gene mutated in ret41 will provide insightful information of the involvement of ethylene in different stages of plant development.
關鍵字(中) ★ 乙烯
★ 小分子化合物
★ 基因功能活化法
★ 化學遺傳法
關鍵字(英) ★ chemical compound
★ activation tagging
★ ethylene
★ chemical genetics
論文目次 目錄
中文摘要………………………………………………………………………….……I
英文摘要…..…………………………………………………………………….…….II
目錄…………………………………………………………………………….……..IV
圖表目錄……………………………………………………………………….……..VI
一、前言……………………………………………………………………….…...….1
二、實驗材料及方法.....................................................................................................8
1. 植物材料及生長方式........................................................................................8
2. 利用小分子化合物篩選出ret突變株..............................................................9
3. 鑑定ret突變株之顯隱性……………………………………………….…...10
4. ret41突變株生理現象之觀察……………………………………………..…10
5. 利用Alexander’s stain方式觀察花粉活性……………………………..…..12
6. 掃描式電子顯微鏡植株樣品之製備……………………………………..…12
7. eto1-5基因型鑑定…………………..………………………………….….…13
8. 南方墨點法………………………………………………………………..…14
9. 鑑定T-DNA 嵌入點………………………………………………….….….17
10. 引子序列…………………………………………………………….…….…19
三、實驗結果…………………………………………………………….…….…….21
篩選ret突變株…………………………………………………………….……21
觀察ret突變株成長狀態………………………………………….…………....22
鑑定ret突變株之顯隱性………………………………………….…………. ..23
鑑定ret41突變株是否為單一或多個突變點…………………….…………....23
分析ret41在培養基中之表現型……………………………….……….……...24
ret41突變株下胚軸長度之分析…………………………………….……….....24
ret41突變株花部組織之觀察………………...…………………………….…..25
ret41突變株雄蕊和花粉粒的活性……………………….………………….…27
eto1-5基因型之鑑定……………………………………………..………….….27
檢測ret41突變株之嵌入點數目…………………………………………….…27
定位T-DNA嵌入點位置…………………………………………………….…28
以基因鑑定確認ret41突變株的T-DNA嵌入點..……………….….………...29
比較ret41突變株和amp1-1、bri1-4、bri1-unknow和bri-201表現型……...30
探討ret41突變株是否牽涉乙烯訊息傳導路徑……………………………….31
ret41突變株和野生型雜交子代的表現型………………………….………….31
製備ret41突變株遺傳圖譜為基礎之基因選殖法之工作….…………………32
四、討論……………………………………………………………………………...33
利用小分子化合物篩選突變庫得到ret突變株…………………………….…33
探討ret突變株之顯隱性………………………………………………….……34
ret41為單一的隱性突變……………………………………..…………………34
探討ret41突變株之生理現象………………………………………….………35
辨別ret41的表現型………………………………………………………….…35
ret41無法得到同型結合子種子的原因…………………………………..……36
探討為何找到的 T-DNA嵌入位置和外表型無法互相吻合……..….…….…37
五、參考文獻…………………………………………………………….………..…39
圖表目錄
圖 一、化學小分子之篩選和利用小分子化合物篩選ret突變株流圖…..…...45
圖 二、小分子化合物的結構圖………………………………………….……..46
圖 三、利用小分子化合物篩選帶有基因功能活化法之ret突變株流程圖….47
圖 四、ret突變株在含有小分子化合物培養基內的表現型……………….….48
圖 五、基因功能活化法ret突變株之成株表現型…………………….………49
圖 六、ret41突變株之成株表現型..................................................................…50
圖 七、EMS ret突變株之成株表現型…………………………………….……52
圖 八、ret突變株之顯、隱性………………………………………….……….53
圖 九、ret41突變株與eto1-5回交後所產生F2子代性狀的比例…...……….54
圖 十、ret41突變株在7303、AVG或STS培養基中之表現型……..……….55
圖 十一、ret41在黑暗或光照下發芽的表現型………..………………………....56
圖 十二、ret41突變株後期成株的表現型………………………………………..58
圖 十三、利用解剖顯微鏡比較ret41突變株和野生型的花部組織……..….…..59
圖 十四、利用電子顯微鏡觀察ret41突變株之花器組織及花粉………….…....61
圖 十五、觀察花粉之活性及測量雄蕊面積…………………………….………..63
圖 十六、利用dCAPS方式確認ret41突變株中帶有eto1-5的突變…….....… .64
圖 十七、利用南方轉漬法分析ret41突變株之T-DNA嵌入點.….…………….65
圖 十八、ret41-5和ret41-23為同一突變株……………………………..……….66
圖 十九、利用TAIL-PCR複製ret41的突變點附近之基因組序列………...…..67
圖 二十、利用質體救援定位嵌入點………………………….……………....…..69
圖二十一、利用基因型鑑定方式確認T-DNA嵌入位置……………………….....70
圖二十二、基因型鑑定結果 …………………………………………………….....71
圖二十三、比較ret41突變株和amp1-1、bri1-4、bri1-unknow和bri-201的幼苗
和成株表現型 ………………………………………………………….72
圖二十四、將ret41突變株和etr1-1、ctr1-1和ein2-1雜交之F1白化苗之表現型
………………………………………………………………………………………...73
圖二十五、ret41突變株和野生種雜交後觀察F1和F2的表現型及eto1-5基因型
定…………………………………………………………………….......74
圖二十六、將ret41突變株和Ler雜交之F1白化苗表現型……….……………..75
附錄 一、基因功能活化法載體-pSKI015………………………………...…..…..76
附錄 二、質體救援法流程…………………………….………………….….……77
附錄 三、TAIL-PCR原理…………………………………………………………78
附錄 四、乙烯生合成及訊息傳導路徑 ……………………………………….…79
附錄 五、ret41和eto1-5回交後子代基因型與表現型之分離釋義圖…………..80
附錄 六、ret41 (F2) 同型結合子和野生型雜交後子代基因型與表現型之分離釋
義圖……………………………………………………………………….81
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指導教授 吳少傑(Shaw-Jye Wu) 審核日期 2011-1-24
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