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博碩士論文 etd-1224111-225021 詳細資訊
Title page for etd-1224111-225021
論文名稱
Title
鎘對不同水稻品種過氧化同功異構酶活性及基因表現之影響
Effects of cadmium on the activity and gene expression of peroxidase isozymes in different Oryza sativa varieties
系所名稱
Department
畢業學年期
Year, semester
語文別
Language
學位類別
Degree
頁數
Number of pages
181
研究生
Author
指導教授
Advisor
召集委員
Convenor
口試委員
Advisory Committee
口試日期
Date of Exam
2011-11-05
繳交日期
Date of Submission
2011-12-24
關鍵字
Keywords
過氧化酶、基因表現、啟動子、鎘、水稻
cadmium, Oryza sativa, gene expression, promoter, peroxidase
統計
Statistics
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The thesis/dissertation has been browsed 5710 times, has been downloaded 2201 times.
中文摘要
鎘為農地主要的污染物之一,嚴重威脅農業生產與為害人類健康,因此植物具鎘耐受性特性品種的栽培與研究特別重要。本論文研究的主要目標在探討對鎘耐受性高的水稻品種之耐鎘機制,針對過氧化酶(POXs)活性變化與基因表現的關係,基因啟動子調控區域內可能的調控元素以及POXs 基因結構分析並探討其親緣關係。試驗以9 種陸稻(水稻另一生態栽培型)與32 種現有水稻栽培品種為材料,利用50 μM CdCl2 濃度處理,探討對水稻種子發芽與幼苗生長的影響,並依其毒害程度,篩選稉稻與秈稻中較耐鎘及敏感品種各2 種,供後續深入研究。結果顯示,陸稻種子發芽受鎘抑制最明顯,其次為秈稻,稉稻則對鎘較具耐受性,顯示水稻品種間有基因型差異。鎘處理除直接抑制種子發芽外,幼根與幼苗伸長明顯受抑制、幼根尖端褐化、根數與根毛的生長均被抑制。在水稻3 葉齡(約12 天)幼苗生長階段,鎘影響幼苗根與莖的伸長,陸稻明顯受鎘抑制,秈稻與稉稻則有不同程度的抑制。因此,水稻種子發芽與幼苗生長受鎘的影響,以發芽階段影響較明顯。依鎘毒害程度,選取稉稻2 個品種,較耐鎘的台農67 號(TNG 67)與較敏感的台農71 號(TNG 71);秈稻2 個品種,秈稻較敏感的台中在來1 號(TN 1)與較耐鎘的台中秈10 號(TCS 10),探討較高鎘濃度(500 μM CdCl2)處理對種子發芽與幼苗生長的影響。結果顯示,稉稻與秈稻品種之種子發芽及12 天幼苗鎘處理對幼根的生長均明顯抑制,對芽鞘的生長抑制則較輕微,高濃度鎘處理有明顯耐受性差異存在。進一步分析品種間過氧化酶活性差異,鎘耐受性較高的水稻品種根部有較高的過氧化酶活性,稉稻亦較秈稻根部有較高的過氧化酶活性,鎘處理明顯改變過氧化酶活性。過氧化酶等電點聚焦電泳條帶存在品種間差異性,所以,鎘處理在不同稻型(稉稻與秈稻)、品種與植株部位(根、芽鞘與葉),對過氧化酶之種類與活性表現,有不同程度的誘導影響。品種間也因不同過氧化酶活化表現與活性改變,產生對鎘之耐受性差異。因此,本研究進一步搜尋水稻資
料庫及選殖出2 個秈稻過氧化酶基因;即pI 4.5 POX 與pI 5.1 POX 基因啟動子(promoter)序列,長度分別為1,225 與1,138 bp,此二序列經PLACE 與PlantCARE
資料庫分析比對轉錄起始位置上游約1,300 bp,結果顯示存在有一些可能參與調控的順式調控元素(cis-acting elements)。這些調控元素可分為三大類:1. 轉錄相關;2. 光調節相關;3. 植物荷爾蒙與逆境相關。通常可藉由基因啟動子序列分析其序列中所含的較短保守序列,得知許多不同種類的轉錄因子(TFs),可能參與基因
表現的調控。2 個過氧化酶基因啟動子序列中均可發現存有與重金屬銅有關之調控元素(CURECORECR),且稉稻與秈稻有個數上差異。另外,所有陸生植物的過
氧化酶基因推測均來自同一祖先,但其基因上游之啟動子序列均有很大的變異,因此,個別的過氧化同功異構酶均有其特殊生理功能。基於水稻與阿拉伯芥過氧化酶之同源關係,本研究另利用6 個阿拉伯芥對鎘處理有明顯活性變化的過氧化酶蛋白質序列為起始點,進行水稻資料庫(RGAP)搜尋比對(BLAST),搜尋所有水稻138 個過氧化同功異構酶中,可能對鎘處理能顯現活性變化的過氧化酶基因。經搜尋發現有9 個分散於2 條染色體上(Ⅲ與Ⅶ)的水稻過氧化酶基因其同源關係特別密切,且分屬2 個基因群集。因此,進一步利用水稻資料庫搜尋、分析與整理這些水稻過氧化酶基因的結構與特性,並探討基因間可能之親緣關係,以及經鎘處理後,不同水稻過氧化酶基因對鎘反應之基因表現情形。試驗結果顯示,這些經搜尋比對而得的水稻過氧化酶基因對鎘處理有明顯之基因表現差異,且其啟動子序列中均可發現存有與重金屬銅有關之調控元素,也有個數上的差異,因此,是否與重金屬鎘的耐受性有關,則有待更進一步研究加以證明。
Abstract
Cadmium (Cd) is one of the major contaminants in agricultural soil, threatening agricultural production and human health. The objectives of this research work were to understand the tolerance mechanism in rice (Oryza sativa L.) genotype with more Cd-tolerance, and the relation between changes of peroxidases activities and peroxidase gene expression profiles after Cd-treated. For more, we analysis about cis-acting elements in the rice peroxidase genes promoter sequences region, gene structure of rice peroxidase genes and phylogenic relation among 9 peroxidase genes which were blasted from 6 Arabidopsis thaliana Cd-induced based on the peroxidase genes protein sequences. We used 9 upland and 32 varieties rice seeds as materials for germination and the growth of seedlings test with 50 or 500 μM CdCl2 application, respectively. Rice seeds germination is a complex physiological and biochemical process, and is highly affected by cadmium. The results showed that Cd inhibited both the growth of radicles and coleoptiles. At germination stage, Cd highly inhibited the growth of upland rice. Among these rice varieties, Japonica type cultivars are more tolerant to Cd, but Indica type are more sensitive to Cd. Upland rice cultivars are the most sensitive to Cd. At seedling stage, Cd highly inhibited the growth of roots, but slightly inhibited the growth of shoots. To cope with Cd-induced stresses, plants adopt different strategies and posses a variety of defense mechanisms to prevent themselves from Cd damage. Peroxidase (POXs) is an important antioxidative enzyme for defense responses against Cd oxidative stress. The results suggested that different rice variety has a specific peroxidase gene expression pattern by the pI focusing electrophoresis after Cd-treated, and the peroxidase activities are significant differences when these rice varieties faced Cd stress. In this study, we searched the rice databases (japonica type) and cloned the promoter sequences of the indica type of the rice peroxidase genes, pI 4.5 POX and pI 5.1 POX genes. According to the search results about cis-acting elements from PLACE and PlantCARE databases, these cis-acting elements can be divided into three classes, showing as follow:1. Transcriptional related; 2. Light regulated related, and 3. Plant hormones- and stress-related. Based on all reported cis-acting elements, there are many types of transcription factors (TFs) involved in regulation of rice pI 4.5 POX and pI 5.1 POX genes expression. These TFs, which can recognize the specific cis-acting elements to regulate the gene expression, and will be induced by stresses and defense-related plant hormones. We found a cis-acting element (CURECORECR) which response to copper in both rice peroxidase genes promoter regions. There is a number of difference between Japonica type and Indica type peroxidase genes, japonica type peroxidase has more cis-acting elements than indica type. Plant class III peroxidases are present in all land plants. All land plant peroxidase genes are with the same putative ancestor of peroxidase genes and are orthologous genes, but they have specific functions of individual perxidase genes owing to their promoter sequences are very divergent. We used 6 Arabidopsis Cd-related peroxidases protein sequences as a starting point for rice peroxidase datas mining. We found 9 rice peroxidase genes have a closely relation among them. For more details about these rice peroxidase genes, searching each one of these rice peroxidases its gene structure on the Rice Genome Annotation Project (RGAP), comparison and their relation. The expressions of these peroxidase genes are very different among them after Cd treatment, and we also found the same cis-acting element (CURECORECR) which response to copper in all 9 rice peroxidase genes promoter regions. There is a number of difference among them.
目次 Table of Contents
誌 謝…………………………………………………………………………… i
中文摘要……………………………………………………………………… ii
英文摘要……………………………………………………………………… iv
目錄…………………………………………………………………………… vi
圖次…………………………………………………………………………… viii
表次…………………………………………………………………………… x
第一章 總論…………………………………………………………………… 1
前言………………………………………………………………………… 1
研究背景…………………………………………………………………… 3
研究方向與實驗架構……………………………………………………… 14
第二章 台灣現有水稻品種(系)對鎘耐受性分析…………………………… 15
中文摘要…………………………………………………………………… 15
英文摘要…………………………………………………………………… 16
前言………………………………………………………………………… 17
材料與方法………………………………………………………………… 19
結果………………………………………………………………………… 21
討論………………………………………………………………………… 25
第三章 鎘對不同水稻品種過氧化酶活性的影響…………………………… 43
中文摘要…………………………………………………………………… 43
英文摘要…………………………………………………………………… 44
前言………………………………………………………………………… 45
材料與方法………………………………………………………………… 48
結果………………………………………………………………………… 50
討論………………………………………………………………………… 56
第四章 不同水稻過氧化酶基因啟動子的研究……………………………… 74
中文摘要…………………………………………………………………… 74
英文摘要…………………………………………………………………… 75
前言………………………………………………………………………… 76
材料與方法………………………………………………………………… 79
結果………………………………………………………………………… 82
討論………………………………………………………………………… 86
第五章 鎘對水稻過氧化同功異構酶基因表現之影響……………………… 114
中文摘要…………………………………………………………………… 114
英文摘要…………………………………………………………………… 115
前言………………………………………………………………………… 116
材料與方法………………………………………………………………… 118
結果………………………………………………………………………… 121
討論………………………………………………………………………… 126
參考文獻……………………………………………………………………… 148
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