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

參考文獻

Betty Chung., Cas Simons., Andrew E Firth1., Chris M Brown1 and Roger P
Hellens (2006). Effect of 5'UTR introns on gene expression in Arabidopsis
thaliana.BMC Genomics 7:1471-2164.
Chai-Fong Lee, Hsin-Yi Pu, Lian-Chin Wang, Ronald J. Sayler, Ching-Hui Yeh,
Shaw-Jye Wu (2006). Mutation in a homolog of yeast Vps53p accounts for the
heat and osmotic hypersensitive phenotypes in Arabidopsis hit1-1 mutant.Planta
224: 330–338.
Eglantine Lobstein2, Anouchka Guyon, Madina Fe´rault, David Twell, Georges
Pelletier, and Sandrine Bonhomme (2004). The Putative Arabidopsis Homolog
of Yeast Vps52p Is Required for Pollen Tube Elongation, Localizes to Golgi, and
Might Be Involved in Vesicle Trafficking. Plant Physiology135:1480–1490.
Elizabeth Conibear and Tom H. Stevens (2000). Vps52p, Vps53p, and Vps54p
Form a Novel Multisubunit Complex Required for Protein Sorting at the Yeast Late
Golgi. Molecular Biology of the Cell 11:305–323.
Elina Welchen, Ivana L. Viola1, Hye Jin Kim, Luciana P. Prendes, Rau´ l N.
Comelli, Jong Chan Hong and Daniel H. Gonzalez (2008). A segment
containing a G-box and an ACGT motif confers differential expression
characteristics and responses to the Arabidopsis Cytc-2 gene, encoding an isoform
of cytochrome c. Journal of Experimental Botany, Vol. 60：829–845.
Elizabeth Conibear, Jessica N. Cleck, and Tom H. Stevens (1999). Vps51p
Mediates the Association of the GARP (Vps52/53/54) Complex with the Late
Golgi t-SNARE Tlg1p. Molecular Biology of the Cell 14：1610–1623.
Georg Jander, Susan R. Norris, Steven D. Rounsley, David F. Bush, Irena M.
Levin1, and Robert L. Last (2002) ..Arabidopsis Map-Based Cloning in the
Post-Genome Era.Plant Physiol.129:440-450
James R. C, Whyte and Sean Munro (2002). Vesicle tethering complexes in
membrane traffic. Journal of Cell Science 115:2627-2637.
Jane Larkindale, Jennifer D. Hall, Marc R. Knight, and Elizabeth Vierling
(2005). Heat Stress Phenotypes of Arabidopsis Mutants Implicate Multiple
~ 32 ~
Signaling Pathways in the Acquisition of Thermotolerance. Plant Physiology
138:882–897.
Jianli Lu, Elumalai Sivamani, Kasi Azhakanandam, Partha Samadder.,
Xianggan Li., Rongda Qu (2007). Gene expression enhancement mediated by the
5’UTR intron of the rice rubi3 gene varied remarkably among tissues in transgenic
rice plants. Mol Genet Genomics 8 :333-6.
Keming Luo, Wei Deng, Shan Xu (2008).Functional analysis of the Arabidopsis
thaliana poly(A) binding protein PAB5 gene promoter in Nicotiana tabacum. Plant
Cell 27:1811-1819.
Lian-Chin Wang, Chin-Hui Yeh, Ronald J. Sayler, Ya-Yun Lee, Chung-An Lu,
Shaw-Jye Wu (2008).Arabidopsis HIT1, a putative homolog of yeast tethering
protein Vps53p, is required for pollen tube elongation.Botanical studies 49:25-32.
Raymond Jozef Maurinus Hulzink, Han Weerdesteyn, Anton Felix Croes, Tom
Gerats, Marinus Maria Antonius van Herpen, and Jacques van Helden (2003).
In Silico Identification of Putative Regulatory Sequence Elements in the
5’Untranslated Region of Genes That Are Expressed during Male Gametogenesis.
Plant Physiology 132: 75–83,
Shiyou Lu¨ A, Hongya Gu A, Xiaojing Yuan, Xiaoming Wang, Ai-Min Wu, Lijia
Qu, Jin-Yuan Liu.(2007). The GUS reporter-aided analysis of the promoter
activities of a rice metallothionein gene reveals different regulatory regions
responsible for tissue-specific and inducible expression in transgenic Arabidopsis.
Transgenic Res16:177–191
Vikrant Gupta, Reema Khurana, Akhilesh K. Tyagi (2007). Promoters of two
anther-specific genes confer organ-specific gene expression in a stage-specific
manner in transgenic systems. Plant Cell Rep26:1919–1931
Wangxia Wang, Basia Vinocur, Arie Altman (2003). Plant responses to drought,
salinity and extreme temperatures:towards genetic engineering for stress tolerance.
Planta 218: 1–14.
A. Wahid , S. Gelani, M. Ashraf, M.R. Foolad (2007).Heat tolerance in plants: An
overview. Environmental and Experimental Botany 61：199–223.
Yu-Tao Yang, Yan-Li Yu, Guo-Dong Yang, Jie-Dao Zhang, and Cheng-Chao
~ 33 ~
Zheng.(2009). Tissue-specific expression of the PNZIP promoter is mediated by
combinatorial interaction of different cis-elements and a novel transcriptional factor.
Nucleic Acids Research: 1–15
Zhihong Lang1, Peng Zhou, Jingjuan Yu1, Guangming and Qian Zhao
(2008).Functional characterization of the pollen-specific SBgLR promoter from
potato (Solanum tuberosum L.).Planta 227:387-396.
Zongrang Liu, Zhongchi Liu (2008). The second intron of AGAMOUS drives
carpeland stamen-specific expression sufficient to induce complete sterility in
Arabidopsis. Plant Cell Rep 27:855–863.

指導教授

吳少傑(Shaw-jye Wu)

審核日期

2009-10-29

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