阿拉伯芥hit3遺傳位址定位與HIT1啟動子分析

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李中瑞(Chung-jui Lee) 查詢紙本館藏

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生命科學系

論文名稱

阿拉伯芥hit3遺傳位址定位與HIT1啟動子分析
(Genetic analysis of heat intolerance3 (hit3) locus inArabidopsis and HIT1 promoter assay)

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摘要(中)

本實驗室之前篩選出用E M S ( e t h y l m e t h a n e s u l f o n i c a c i d )化學突變劑，所產生不具有耐熱特性的阿拉伯芥突變植株，命名為h i t 1 - 1 (h e a t i nt o l e r a n c e ) 。經由分析後得知突變基因位於阿拉伯芥第一條染色體上，且其基因所編碼之蛋白質與酵母菌(S a c c h a r o m y c e s c e r e v i s i a e )之V p s 5 3 p 相似。為了更了解H I T 1
基因在植物組織中分佈的情形，將H I T 1 基因之啟動子全長約
2 . 4 K b 與β - g l u c u r o n i d a s ( G U S )報導基因相連，觀察H I T 1 在植物中表現情形，發現H I T 1 在已成熟的花粉、花粉管、及根尖伸長區的地方有表現，顯示H I T 1 基因具有組織特異性。在序列分析後發現啟動子上具有兩組a n t h e r / p o l l e n - s p e c i f i c e l e m e n t
G T G A 及A G A A A，因此推測此e l e m e n t 或許是造成H I T 1 主要表現在根尖及花粉管之因。此外，利用5’ d e l e t e m u t a t i o n 研究H I T 1啟動子，發現H I T 1 - D 3 d e l e t i o n 及H I T 1 - D - U T R d e l e t i o n 會導
致基因無法表現。經由序列比對，發現H I T 1 - D 3 片段不具有G - b o x e l e m e n t 。G - b o x 與光感應有關，若干研究也曾指出刪除啟動子的G - b o x 片段會影響基因的表現，因此推測H I T 1 - D 3 基因之不表現可能是因為G - b o x e l e m e n t 的喪失。此外， 5’ U T R 為起始密
碼子A T G 前一段非編碼區。在d e l e t i o n 後導致基因不表現，推測可能是後轉譯或轉錄受影響。此外，本實驗室利用相同方法篩選出另一突變株h i t 3 。利用基因定位法研究h i t 3 突變位址，目前推測在阿拉伯芥第五條染色體。進一步研究h i t 3 將有助於對植物耐熱機制的了解。

摘要(英)

Previously, in order to understand the mechanisms of plant tolerance, we used chemical mutagen EMS to generate point mutation and isolated a heat intolerant mutant from Arabidopsis which is named hit1. HIT1 locus was later identified to be on the chromosome Ι and amino acid analysis showed that HIT1 is homologous to yeast Vps53p. In order to understand the physiological function of HIT1, we use HIT1 promoter driven -glucuromidase (GUS) reporter gene to observe the expression pattern in plant. Result showed that HIT1 expression only at elongation zone of root,stigma and pollen tube. DNA sequence analysis further indicated that there are two anther/pollen-specific elements GTGA and AGAAA present in HIT1 promoter. It
might be the cause of the observed organ/tissue expression specificity. Moreover, in order to dissect the regions that lead to the HIT1 expression specificity, a series of
5’-deletion of HIT1 promoter were generated by PCR from Columbia (WT) to drive GUS reporter gene. Results indicated that HIT1-D3 might have important element that
deletion of such region can inhibit HIT1 gene expression. Sequence analysis also showed a G-box which may be an important regulatory element of the gene. The 5’’ untranslated region also showed effects on HIT1 gene expression, possibly through post- transcriptional regulation. In the mean time, another EMS-mutagenized heat
intolerant mutant, hit3, was isolated from Arabidopsis. The locus of hit3 currently mapped to chromosome Ⅴ. More genetic analysis and physiological test of the hit3
mutant should provide insight to the complexity of plant heat tolerance.

關鍵字(中)

★ 熱處理
★ 阿拉伯芥
★ 啟動子分析
★ 基因定位

關鍵字(英)

★ promoter assay
★ mapping
★ heat stress
★ Arabidopsis

論文目次

目錄
壹、前言 1
貳、材料與方法 9
一 HIT1基因在植物體中表現位置 9
1.primer(引子)設計 9
2.載體DNA之製備 10
3.接合反應 11
4.細菌轉殖 12
5.細菌質體DNA小量(Mini)抽法 12
6.細菌質體DNA小量(Mini)製備 13
7.農桿菌(GV1301)轉植(Agro transformation) 14
8. GUS染色 15
9. GUS活性測試 15
二 HIT3基因之基因定位 17
1.種子之製備 17
2.配置培養基 17
3. 種子清洗 17
4. 植株熱處理 18
5. 阿拉伯芥DNA之萃取 18
6. 利用基因標記定位出hit3的突變位址 19
三 HIT3基因生理實驗 20
1.鹽類濃度試驗 20
2.Heat shock生理實驗 21
参、結果與討論 22
1. HIT1基因在植物組織中之表現情形 22
2. HIT1啟動子GUS活性測試 27
3.以遺傳圖譜為基礎進行HIT3基因定位 27
4.HIT3基因之生理試驗 29
肆、參考文獻 30
圖表目錄
圖 1 HIT1啟動子與基因體質圖示及專一性引子 36
圖 2 利用PCR分析轉植株 37
圖 3 HIT1基因在植物組織中表現情形 42
圖4HIT1啟動子GUS活性測試 44
圖 5 HIT3突變株在37℃下處理5天後之型態 45
圖 6 HIT3突變株在經由高溫處理下與野生型外觀之差異 46
圖 7 HIT3與分子標記間的交換率 47
圖 8 野生型、HIT1及HIT3在不同鹽分濃度下其生長型態 49
圖 9野生型、HIT1及HIT3對heat shock後之反應 50
表 1 HIT1 啟動子cis-acting regulatory分析 52
表 2 HIT3與染色體間分離情形所需之分子標記之引子序列 53
附錄 1 PCR引子列表 54
附錄 2 66
附錄 3 69

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指導教授

吳少傑(Shaw-jye Wu)

審核日期

2009-10-29

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