利用cDNA微陣列技術探討蕃茄根部組織受過量銅誘導之基因

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楊明華(Ming-Hua Yang) 查詢紙本館藏

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生命科學系

論文名稱

利用cDNA微陣列技術探討蕃茄根部組織受過量銅誘導之基因
(Functional Genomics Study in the Roots of Tomato (Lycopersicon escolentum) in Response to Excess of Copper Using cDNA Microarray Technique)

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摘要(中)

中文摘要
對植物而言，銅是一必需的微量元素。在植物細胞內，銅可作為許多參與氧化反應、光合作用酵素的輔助因子。然而，過量銅存在會引起毒性反應如氧化性之壓迫，導致植物矮小以及死亡。另一方面，在缺乏銅的環境下，葉子會有彎曲變形以及提早老化的病徵。根是植物吸收土壤中金屬微量元素的重要組織器官，因此根部細胞可說是擔任控制銅離子恆定作用的第一道前線。然而目前根部細胞內銅離子恆定機制及生理功能並不清楚，所以本論文是採用功能性基因組之研究探討蕃茄根部組織在過量銅環境下誘導的特定基因，由這些基因已知的生理作用及相關途徑，來說明其在植物根部組織中銅離子恆定作用的機制。而我主要的實驗是利用差異式基因選殖與cDNA微陣列技術來大量收集受過量銅誘導的基因，且獲得其核苷酸序列並利用基因資料庫作序列的比對推斷其可能的身分。目前已從10 mM CuSO4處理四天之蕃茄根部細胞的相減式cDNA基因庫中，挑出48個受銅誘導兩倍以上的基因，而以基因的生理作用可分類為：（一）與抗氧化逆境相關之基因；（二）與抗病源體感染相關之基因；（三）與賀爾蒙的合成相關之基因；（四）與細胞的修復相關之基因；（五）其它基因群。

摘要(英)

Abstract
Copper is an essential trace element in plants. It is a cofactor for many cellular enzymes involving oxidation and photosynthesis. Excess amount of copper causes oxidative stress in cells and may lead stunted growth or death. On the other hand, the deficiency of copper causes several symptoms such as distortion and premature senescence of young leaves. Plants acquire trace elements such as copper through root system. Thus, root tissue stays in the front line and plays a key role in regulating copper homeostasis. However, the physiological mechanism of controlling copper concentration in root is still unclear. This thesis adopts functional genomic approach to unfold specific gene expression in various physiological processes under the treatment of excess copper to tomato root tissue. The up-regulated genes may help to illustrate the partial scheme of copper homeostasis in root system. The differential cloning method is employed to select copper-induced clones. Subsequently, the copper responsive genes were collected by cDNA microarray. The identities may be revealed by DNA sequencing and by the alignment of the gene sequences in Gene Bank. Forty-eight up-regulated clones of over two folds were identified from the cDNA subtraction library that was generated from the continuous treatment of 10 μM copper sulfate for 4 days in tomato roots. Based on their physiological implication, they can be grouped into five categories: (1) anti-oxidation (2) pathogen response (3) hormone biosynthesis (4) cellular organization (5) others.

關鍵字(中)

★ 蕃茄
★ 重金屬
★ 銅離子恆定機制
★ 相減式cDNA雜交法
★ cDNA微陣列
★ 功能性基因組

關鍵字(英)

★ cDNA microarray
★ subtraction cDNA hybridization
★ copper homeostasis
★ heavy metal
★ tomato
★ functional genomics

論文目次

目錄
中文摘要--------------------------------------------------------- Ⅰ
英文摘要--------------------------------------------------------- Ⅱ
目錄--------------------------------------------------------------- Ⅲ
『圖』目錄------------------------------------------------------ Ⅵ
『表』目錄------------------------------------------------------ Ⅶ
縮寫表--------------------------------------------- --------------- Ⅷ
第一章緒論
壹、在植物中金屬的恆定機制------------------------ 1
貳、銅的特性及其功能--------------------------------- 3
參、過量銅離子濃度對植物的影響------------------ 4
肆、文獻已知受過量銅誘導的基因------------------ 5
伍、相減式雜交法--------------------------------------- 5
陸、 cDNA微陣列（cDNA microarray）的簡介--- 6
柒、研究動機--------------------------------------------- 7
捌、研究目的--------------------------------------------- 8
第二章材料與方法
第一節實驗材料
壹、種子的來源------------------------------------------ 10
貳、蕃茄的種植------------------------------------------ 10
第二節實驗方法
壹、不同銅離子濃度及處理時間點的毒性測驗--- 11
貳、銅在植物體內的累積量之測定------------------- 11
參、北方點墨法------------------------------------------- 12
肆、訊息核糖核酸（mRNA）之純化與分析-------- 14
伍、相減式cDNA雜交法------------------------------- 16
陸、建立相減式cDNA基因庫------------------------- 21
柒、 cDNA微陣列----------------------------------------- 23
捌、基因定序與分析------------------------------------- 27
第三章實驗結果
第一節過量銅濃度對蕃茄生長的影響
壹、過量銅濃度對蕃茄之外觀型態的影響----------- 29
貳、過量銅濃度對蕃茄根部細胞內標的基因的影響
參、銅在shoot與root的累積量----------------------- 29
第二節建立相減式cDNA基因庫
壹、訊息核糖核酸（mRNA）之純化與分析------- 30
貳、相減式cDNA雜交法所得到可能受過量銅誘導
的基因------------------------------------------------- 31
第三節 cDNA微陣分析誘導基因之表現
壹、相減式cDNA基因庫之誘導基因確立---------- 32
貳、探討在蕃茄根部細胞內受過量銅誘導的基因
表現情形---------------------------------------------- 32
參、 DNA定序及基因鑑定------------------------------ 33
第四章討論
第一節　受過量銅誘導基因之探討------------------------- 34
第二節　受過量銅誘導基因與其它逆境的關係---------- 40
第五章結論與建議
壹、結論---------------------------------------------------- 42
貳、建議 ---------------------------------------------------- 43
參考文獻---------------------------------------------------------- 45
附錄 --------------------------------------------------------------- 82
圖目錄
圖一、在植物中金屬恆定機制的假設系統圖------------------------ 52
圖二、目前已知的植物金屬運輸蛋白（transpoters）-------------- 53
圖三、製備相減式cDNA雜交法實驗流程--------------------------- 54
圖四-1、蕃茄幼苗經毒性測驗4天後之外觀型態變化------------- 55
圖四-2、蕃茄幼苗經毒性測驗4天後之外觀型態變化（近照）-- 56
圖五、北方點墨法分析蕃茄根部LEMT1基因------------------------- 57
圖六、蕃茄根部的總核糖核酸（total RNA）純化與分析---------- 58
圖七、蕃茄根部的訊息核糖核酸（mRNA）純化與分析---------- 59
圖八、確定過量銅之相減式cDNA雜交結果------------------------ 60
圖九、受過量銅誘導蕃茄根部細胞的基因片段之確定------------ 61
圖十、cDNA微陣列圖像------------------------------------------------ 62
圖十一、受過量銅誘導的基因表現倍率之分散曲線圖------------ 63
圖十二、受過量銅誘導的基因表達倍數之數目統計--------------- 64
圖十三、與抗病原體有關的訊息傳遞途徑--------------------------- 65
表目錄
表一、Hoagland水耕培養液------------------------------------------- 66
表二、毒性測驗培養的條件----------------------------------------- -- 67
表三、本研究所選用引子（primer）之序列----------------------- 68
表四、銅在植物體內的累積分布情形------------------------------- 69
表五-1、與抗氧化逆境相關之基因---------------------------------- 70
表五-2、與抗病源體感染相關之基因------------------------------- 71
表五-3、與抗病源體感染相關之基因------------------------------- 72
表五-4、與抗病源體感染相關之基因------------------------------- 73
表五-5、與賀爾蒙的合成相關之基因------------------------------- 74
表五-6、與細胞的修復相關之基因---------------------------------- 75
表五-7、其它基因群---------------------------------------------------- 76
表五-8、其它基因群---------------------------------------------------- 77
表五-9、其它基因群---------------------------------------------------- 78
表五-10、其它基因群-------------------------------------------------- 79
表五-11、其它基因群-------------------------------------------------- 80
表六、受過量銅誘導基因對其他逆境之多重反應---------------- 81

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指導教授

董啟功(Chii-Gong Tong)

審核日期

2002-7-18

推文