博碩士論文 91224002 詳細資訊


姓名 蒲欣儀(Shin-Yi Pu)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 阿拉伯芥突變種(hit1)之位址定位
(Genetic Mapping of hit1 Locus in Arabidopsis thaliana)
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摘要(中) 中文摘要
為了了解植物耐受高溫的遺傳控制因子及其生理機制,我們用化學突
變劑( EMS )誘導阿拉伯芥點突變產生,並進而篩選出一株對熱缺乏耐受
性之突變種,稱之為hit1( heat intolerance ) 。這是一種典型從具遺傳性之
生理性狀找出相對應之基因及其分子作用的研究方式,亦即所謂的向前式
遺傳學分析法( forward genetics ),為功能性遺傳學研究法中的一種。因
hit1 之性狀為點突變所造成的,故其功能性基因須以遺傳圖譜為基礎來進
行定位選殖( map-based cloning, MBC ) 。其做法是先將Columbia 生態
型之hit1 突變種與Landsberg 生態型之野生種雜交,之後的F1 自交得F2
世代,F2 世代中帶有hit1 性狀之個體為hit1 定位時所需之分離族群
( segregation population )。以hit1 之突變性狀,37℃處理四天後會死亡的
原則篩選, 再經簡單序列長度多型性( simple sequence length
polymorphism , SSLP )及限制酵素切割擴增片段多型性( cleaved amplified
polymorphic sequence , CAPS )兩種對生態型具專一性之分子標記進行定
位。本研究共設計了26 組SSLP 分子標記,7 組CAPS 分子標記。在檢
測過3987 株F2 世代的hit1 突變種之後,推得hit1 基因坐落在第一條染色
體, AGI map 18767.3 kb ~ 18810.2 kb 之42.9kb 範圍內,達到MBC 所需
之定位目標。
摘要(英) Abstract
In order to understand the mechanism of plant crop heat stress, we
screened a mutant that is difficult to tolerant heat stress called “hit1”
(heat intolerance) inducible by chemical mutagenesis, EMS, causing a
point mutation. Forward genetic is one of functional genetics approaches,
and it is underlying a mutant with a desirable genetic phenotype to an
identified mutation in a gene and to analysis specific gene’s molecular
biology function. We use forward genetic to approach “hit1” that was a
point mutation, so we want a cloning of sequencing mutant-define gene
by map-based coloning (MBC). A hit1 mutant with Columbia ecotype (P1)
and a wild type with Landsberg erect ecotype (P2) were parent to get
recombinant inbred lines (F2). It has hit1 mutant specific phenotype lines
of F2 generation that is a segregation population to provide hit1 genetic
mapping. The definition of hit1 mutant phenotype was death at 37℃ in 4
days to screen mutants from F2 generation as a segregation population
then make hit1 gene genetic mapping, and we designed 26 pairs primers
of simple sequence length polymorphism (SSLP) markers and 7 pairs
primers of cleavage amplify polymorphisms (CAPS) markers for genetic
analysis. We suggest that hit1 gene locus in AGI 18767.3kb ~ 18810.2kb,
total 42.9kb regions, and approach the fine-scale mapping of MBC.
關鍵字(中) ★ 阿拉伯芥
★ 對熱不耐受性突變株
關鍵字(英) ★ hit1
★ heat intolerance
★ Arabidopsis
論文目次 III
目錄
中文摘要……………………………………………………………Ⅰ
英文摘要…………………………………………………………..Ⅱ
目錄…………………………………………………………………Ⅲ
圖目錄…………………………………………………………..…..Ⅶ
表目錄……………………………………………………...……….Ⅷ
縮寫與全名對照表…………………………………………………Ⅸ
第一章序論…………………………….…………...1
壹、阿拉伯芥的簡介………………………………………...1
貳、研究基因的方法………………………………………...2
一、Reverse genetics: ……………………………………..….2
二、Forward genetics: ……………………………………..….3
參、導致突變的方法…………………………………………..4
IV
肆、突變點的定位方法…………………………………..………7
一、以遺傳圖譜為基礎之選殖基因法( map-based cloning, MBC ) ….........7
二、各種標記之簡介: …………………………………………...8
A). 標記之分類: …………………………………………........8
B). DNA 分子標記(DNA molecular marker) ……….…………………9
1). 限制片段長度多型性RFLP (restriction fragment length polymorphism) 11
2). 逢機增殖多型性DNA (random amplified polymorphic DNA, RAPD) ⋯12
3). 擴增片段長度多型性(amplified fragment length polymorphism, AFLP) ..13
4). 簡單序列長度多型性( simple sequence length polymorphism , SSLP ) ....14
5). 限制酵素切割擴增片段多型性( cleaved amplified polymorphic
sequence , CAPS ) ……………………………………….16
三、利用CELΙ 切割技術來輔助突變點的定位………………………17
伍、研究動機與目的………………………………………..……19
第二章材料與方法…………………………………………..22
1. 阿拉伯芥基因體基因之粗萃取…………………………………….........22
2. 製備挑選hit1 surpressor mutant 之M2 種子………………………………..23
3. 挑選hit突變株……………………………………………………….23
4. 挑選surpressor mutation …………………………………………….......24
5. 製備挑選定位(mapping)時所需之hit1種子……………………………….25
6. 挑選定位時所需之hit1突變株………………………………………….25
7. 阿拉伯芥成株之培植…………………………………………………..26
8. 突變種隱、顯性之鑑定………………………………………………...26
9. 功能性對偶基因之互補測試(complementation assay) …………......................26
10. 上位(epistatic) 現象之測試…………………………………………….27
11. 卡方適合性分析(χ2‧goodness-of-fit test) ………………………………...28
12. SSLP 之操作步驟……………………………………………………....28
V
13. CAPS 之操作步驟………………………………………………………30
14. CELI 之操作步驟……………………………………………………….31
15. 設計引子所需考慮的細節…………………………………………….....32
第三章結果……………………………………………………...34
壹、hit1 的生長型態…………………………………………………34
貳、hit1 基因之遺傳分析……………………………………………35
參、hit1 基因之定位…………………………………………………36
一、以37℃ 處理4 天後死亡之突變株(F2) 進行定位………………..37
二、以對熱敏感之hit1 突變株(F2) 進行hit1 進行定位………………37
三、以CEL I 酵素切割來輔助hit1 之定位………………..…………..40
肆、hit 突變株之篩選…………………………………………………41
一、hit1 suppressor mutant …………………………………………….41
二、hit 突變株……………………………………………………....41
第四章結論……………………………………………………43
壹、阿拉伯芥功能性基因之開發……………………………………...43
貳、hit1 基因之定位…………………………………………………....44
參、分子標記SSLP 及CAPS 之使用………………………………...45
肆、以CELΙ 酵素切割來輔助hit1 之定位…………………...............46
VI
伍、hit1 基因之遺傳分析…………………………………..…...48
陸、hit 突變株之篩選……………………………………….....49
第五章結論……………………………………………..52
第六章文獻探討…………………………………………53
圖表……………………………………………………………...….61
附錄…………………………………………………………………82
一、植物培養基之配製法………………………………………….. 82
二、溶液及試劑配方……………………………………………….82
三、藥品試劑………………………………………………………85
四、酵素與限制酶………………………………………………… 86
五、儀器………………………………………………………… 86
六、hit1 基因之cDNA 序列與資料庫(NM_103933/At1g50500)之差異性。…87
七、hit1 基因與多種物種之間保守區間之比對圖。……………………..88
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指導教授 吳少傑(Show-Jye Wu) 審核日期 2004-7-15
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