利用基因功能活化法研究阿拉伯芥乙烯生合成之調控機制

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姓名

劉佳苹(Chia-ping Liu) 查詢紙本館藏

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生命科學系

論文名稱

利用基因功能活化法研究阿拉伯芥乙烯生合成之調控機制
(Studying ethylene biosynthesis in Arabidopsis thaliana by activation tagging approach)

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★ 蛋白質法尼脂化修飾參與植株耐熱反應	★ 探討ETO1-LIKE1(EOL1)及EOL2參與阿拉伯芥幼苗光形態發育之功能

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至系統瀏覽論文 ( 永不開放)

摘要(中)

乙烯是種化學結構簡單的氣體荷爾蒙，在植物的生活史中，從種子萌發，開花受粉，果實成熟，到植物老化，乙烯都扮演重要的角色。乙烯也會和植物體內的其他賀爾蒙 (例如水揚酸，茉莉酸及離層酸) 產生交互作用，讓植物能夠在逆境時產生適當的反應來適應環境的變化而求得生存。乙烯的生合成是SAM (S-adenosylmethionine)經過ACC (1-aminocyclopropane-1-carboxylic acid) 合成酶(ACS) 的催化形成ACC，再經由ACC氧化酶作用而產生。之前研究顯示，在阿拉伯芥中的ETO1 (ETHYLENE OVERPRODUCER1) 產生突變，會造成乙烯大量產生。因此可推論ETO1會負向調控乙烯的生合成。
ETO1屬於一個植物基因家族，包括EOL1及EOL2 (ETO1-LIKE) 共三個成員，帶有BTB (Broad-Complex, Tramtrack, and Bric a brac) 和 TPR (Tetratricopeptide Repeat) 的功能性區域。這兩個區域與蛋白質交互作用有關。本論文的研究是利用基因功能活化法 (activation tagging) 篩選及鑑定eto1-5突變株的抑制基因 (suppressor gene)；藉由篩選基因功能活化法所得到的突變株，找到16個seat (suppressor of eto1-5 by activation tagging) 的突變株，可有效抑制eto1-5性狀 (phenotype)，經由初步之生理及遺傳研究，確定所有seat突變株皆為顯性突變 (dominant mutation)。此外也鑑定這些突變株的T-DNA嵌入點 (insertion site) ，用以分析哪些鄰近基因可能是造成seat表現型態的原因。未來，將製備轉殖基因植物來證實所選擇的基因是否真的會抑制eto1-5的突變性狀。

摘要(英)

Ethylene is a gaseous phytohormone and has a versatile role in plant physiology. Ethylene biosynthesis is committed by the conversion of S-adenosylmethionine (SAM) to 1-aminocyclopropane-1-carboxylic acid (ACC) by ACC synthase (ACS). Previous studies showed that ETO1 (ETHYLENE OVERPRODUCER1) negatively regulated ethylene biosynthesis and resulted in increased ethylene production in Arabidopsis thaliana.
ETO1 represents a gene family in Arabidopsis thaliana, including EOL1 and EOL2 (ETO1-LIKE). There are two distinct protein domain in ETO1. BTB (Broad-Complex, Tramtrack, and Bric a brac) and TPR (Tetratricopeptide Repeat) domains, which both are involved in related with protein-protein interactions. My thesis research is to use activation T-DNA tagging approach to identify suppressors of eto1-5 in Arabidopsis thaliana. By screening the activation tagging mutants carrying an eto1-5 allele, we have found 16 seat (suppressor of eto1-5 by activation tagging) mutant lines. All of the 16 seat mutants are dominant and belong to at least 3 complementation groups. I have identified the T-DNA insertion sites in the seat mutants to analyse how the flanking genes regulate seedling phenotype of eto1-5. In the future work, Arabidopsis transgenic lines will be generated to confirm the function of selected genes as suppressors of eto1-5.

關鍵字(中)

★ 乙烯
★ 基因功能活化法

關鍵字(英)

★ ethylene
★ activation tagging

論文目次

中文摘要……………………………………………………………...I
Abstract …………………………………………………………….II
……………………………………………………………………… III
圖表目錄……………………………………………………………….V
縮寫對照表………………………………………………………….VII
壹、前言…………………………………………………………....-1-
貳、實驗材料…………………………………………………………-6-
I. 植物材料………………………………………………… -6-
II. 基因功能活化法載體－pSKI015………………………. -6-
III. 植物生長培養基.................................-6-
IV. seat突變株篩選………………………………………….-6-
V. 引子的序列.....................................-7-
VI. 緩衝液的配置...................................-9-
參、實驗方法……………………………………………………….-10-
I. 利用基因功能活化法篩選seat突變株 ……………….-10-
II. seat突變株生理現象之觀察..................... -10-
III. seat T3 和T4代進行基因型鑑定……………………..-11-
IV. 鑑定seat突變株之顯隱性.....................................................-12-
V. 鑑定T-DNA 嵌入點..............................-13-
VI. 鑑定T-DNA 嵌入點上下游基因的影響............. -15-
VII. 基因型鑑定確認T-DNA位置…………………………...-17-
VIII. 回交後的seat 突變株進行基因型鑑定……………….-17-
肆、實驗結果…………………………………………………………………….-19-
利用基因功能活化法篩選seat突變株…………………………… -18-
seat 突變株進行基因型鑑定和表現型鑑定………………………-19-
觀察seat 突變株與 eto1-5回交後所產生F1子代的性狀……… -20-定位T-DNA 嵌入點位置.....................................................-21-
基因型鑑定確認T-DNA位置…………………………………………-21-
T-DNA 嵌入點上下游基因表現量的影響………………………… -22-
seat F3突變株生理現象之觀察 ………………………………… -23-
伍、討論…………………………………………………………………… -25-
利用基因功能活化法篩選seat突變株…………………………… -25-
seat 突變株與 eto1-5回交後產生的F1子代性狀……………… -25-
定位T-DNA 嵌入點位置…………………………………………… -25-
基因型鑑定確認T-DNA位置…………………………………………-28-
seat F3突變株生理現象之觀察……………………………………-28-
陸、後記…………………………………………………………… -31-
回交後的seat 突變株進行基因型鑑定……………………………-31-
柒、參考文獻……………………………………………………… -32-

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指導教授

吳少傑、王隆祺
(Shaw-Jye Wu、Long-Chi Wang)

審核日期

2009-10-2

推文