阿拉伯芥突變種hit2之位址定位

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吳欣潔(Shin-jye Wu) 查詢紙本館藏

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生命科學系

論文名稱

阿拉伯芥突變種hit2之位址定位
(Genetic Mapping of hit2 Locus in Arabidopsis thaliana)

相關論文

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摘要(中)

摘要
高溫逆境為主要的環境逆境之一，會抑制植物的生長甚至造成植物死亡。過去對於高溫逆境的研究主要為短暫的高溫逆境，而長時間的高溫逆境較少被人研究，本研究的目的即是以向前式遺傳研究法(forward genetics approach)來找出植物對抗長時間高溫逆境的基因。利用ethylmethane sulfonate為突變劑，我們從阿拉伯芥中篩出一突變株，取名為hit2 (heat intolerance 2)。以37℃熱處理3天後，hit2會呈現白化死亡而野生型則能繼續生長。除此之外，hit2對於heat shock與巴拉刈(paraquat)所造成的逆境也有低耐受的表現型態。我們使用以遺傳圖譜為基礎之基因選殖法(map-based cloning, MBC)，將Columbia生態型之hit2突變株與Landsberg erecta生態型之野生種雜交，其自交之F2子代再以基因標記進行基因定位分析。在檢測了2675棵F2植株後，我們發現hit2突變點的位址坐落於阿拉伯芥第五條染色體AGI map 5,570kb與5,620kb之間。在進行核甘酸定序後我們在一個NADPH依賴的氧化還原酶(NADPH-dependent oxidoreductase)基因中找到一個由C變T的點突變。此外，我們也比較了hit2與先前篩選出之另一個對熱耐受性較低的突變株hit1-1對不同逆境之反應。根據胺基酸的序列比對，HIT1與囊泡繫鏈蛋白質(vesicle tethering protein)同源。而實驗的結果則顯示，巴拉刈會影響hit1-1的生長，但以heat shock處理後之hit1-1則與野生型一樣能繼續生長。據此，我們推測HIT1與HIT2以不同的機制來幫助植物對抗高溫逆境。HIT1有可能是以囊泡循環來幫助植物細胞修復受損的細胞膜，而HIT2則是在負責降解熱逆境所產生的過氧化物所造成的有毒物質。接下來，我們會將野生型的HIT2基因轉殖到hit2突變株進行互補試驗，並研究HIT2在植物生理中擔任的功能與角色。

摘要(英)

ABSTRACT
Heat stress as a result of elevated temperatures is one of the major agricultural problems that limits crop yield. To understand how plants respond to survive such detrimental condition and what genetic determinants are responsible for the protecting responses, forward genetic approach was employed to directly identify genes that play active roles in plant heat tolerance. Arabidopsis hit2 mutant (as heat-intolerance 2) was hence identified through screening of ethylmethane sulfonate (EMS)-mutagenized M2 plants. The growth of hit2 mutant is more sensitive than that of wild type plants under non-lethal, prolonged high temperature stress (37oC for 3 days). In addition, hit2 mutant is hypersensitive to both heat shock and methyl viologen (MV, also known as paraquat) treatments. For gene mapping, mutants in the Columbia background were crossed to plants of the Landsberg erecta background and F1 plants were allowed self-pollinated to produce the F2 plants. About 2700 F2 individuals with hit2 phenotype were collected and subjected to the mapping analysis. Results indicated that hit2 is located at the region between AGI map 5,570kb and 5,620kb of the chromosome 5, which is covered by bacterial artificial chromosome F2K13. Sequencing of the entire region revealed a single nucleotide substitution from C to T in a gene encoding a putative NADPH dependent oxidoreductase. We also compare the stress sensitivity between hit2 and hit1-1, a previously identified heat intolerant mutant whose mutated gene product is homologues to yeast tethering protein Vps53p. Results showed that hit1-1 mutant is hypersensitive to paraquat treatment but resistant to heat shock treatment. Accordingly, we proposed that HIT1 and HIT2 protect plants from heat stress damage via different mechanisms. It is suggested that HIT1 plays the role in repairing damaged membrane through vesicle recycling while HIT2 is responsible for protecting plants from high temperature induced oxidative stress. Currently, we are cloning the wild-type HIT2 gene for complementation test. Examination of the Molecular and cellular function of HIT2 will follow as well.

關鍵字(中)

★ 對熱耐受性較低的突變株
★ 氧化還原酶
★ 阿拉伯芥

關鍵字(英)

★ oxidoreductase
★ heat intolerance
★ hit2
★ Arabidopsis

論文目次

目錄
中文摘要------------------------------I
英文摘要-----------------------------II
致謝--------------------------------III
目錄---------------------------------IV
圖表目錄-----------------------------VI
緒論----------------------------------1
材料與方法----------------------------6
1. hit2突變株之顯隱性鑑定--------6
2. hit2突變株之位址定位----------6
(1) hit2位址定位之種子製備--------6
(2) 挑選定位所需的hit2突變株------7
(3) hit2突變點之位址定位----------8
(4) F3表現型分析-----------------12
3. 核甘酸之定序-----------------12
4. HIT2基因之質體構築-----------14
(1) mRNA之萃取-------------------14
(2) cDNA合成---------------------15
(3) 以PCR擴增HIT2基因------------16
(4) DNA接合反應------------------17
(5) 勝任細胞之製作---------------17
(6) 細菌轉殖---------------------18
(7) 質體DNA之萃取----------------18
(8) HIT2基因轉殖確認-------------19
5. hit1、hit2突變株處理巴拉刈表現型測試--20
6. hit1、hit2突變株處理heat shock表現型測試--20
結果---------------------------------21
1. hit2突變株之表現型-----------21
2. hit2突變株之顯隱性鑑定-------21
3. hit2突變點之位址定位---------21
4. 核甘酸定序尋找突變點---------23
5. HIT2基因之質體構築-----------23
6. 巴拉刈表現型測試-------------24
7. Heat shock表現型測試---------25
討論---------------------------------26
1. HIT2基因的功能---------------26
2. 生理試驗---------------------29
參考資料-----------------------------34
圖表目錄
圖一、hit2突變株與野生型表現型比較---40
圖二、hit2突變株定位之F2表現型挑選---41
圖三、hit2突變株表現型---------------42
圖四、hit2與Col雜交產生之 F1表現型測試-43
圖五、不經過熱處理之F2分離情形--------44
圖六、C483050與C483070定序結果--------45
圖七、hit2突變株位址定位結果----------46
圖八、核甘酸定序結果------------------47
圖九、HIT2基因------------------------48
圖十、hit1與hit2處理巴拉刈之種子發芽--49
圖十一、hit1與hit2處理巴拉刈之幼苗成熟-50
圖十二、 hit1與hit2 37 ℃熱處理表現型--51
圖十三、hit1與hit2 heat shock處理表現型-52
圖十四、HIT1、HIT2在植物對抗環境逆境的角色-53
表一、hit2突變株之顯隱性鑑定-------------54
表二、不經過熱處理之F2分離情形-----------55
表三、經過熱處理之F2位址分析數據---------57
附件一、位址定位之引子-------------------58
附件二、定序之引子-----------------------59
附件三、At5g16790在阿拉伯芥發育表現情形--64

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指導教授

吳少傑(Shaw-jye Wu)

審核日期

2009-7-16

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