蕃茄根部受銅逆境之基因調控

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姓名

黃雅琳(Ya-Ling Huang) 查詢紙本館藏

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生命科學系

論文名稱

蕃茄根部受銅逆境之基因調控
(Gene regulation of copper stress in Lycopersicon esculentum (cv CL5915) root tissue)

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摘要(中)

銅對於植物的生長為一必須的微量金屬元素，其對許多酵素之功能上為一無可替代的輔因子。過量銅濃度下，在生物體內會造成Fenton反應，促使活性氧化物生成，進一步對生物體造成傷害。為了避免傷害的造成，植物體中必然有解毒機制存在。直至目前為止，尚未有針對於過量銅對植物體基因表現上造成的改變之研究報告。在本論文中，使用反向相減式微陣列、中研院生農所蕃茄基因微陣列及康乃爾蕃茄基因微陣列，篩選受過量銅影響表現之基因。我們發現在過量銅處理下，共有22個無重複之基因(tentative consensus)受到抑制，而有72個基因受到誘導。在銅逆境下會誘導大部分的ROS清除者，包含glutathione S-transferase/peroxidase、ascorbate free radical reductase及cytochrome P450，顯示銅逆境與氧化性傷害具有高度之相關性。除此之外heat shock proteins也受到過量銅的誘導，象徵對銅逆境的耐受性增加。而Tonoplast instrinsic protein delta type及vacuolar H+ ATPase B subunit在銅逆境下則受到抑制，這可能表示液泡在受過量銅處理下會有所變化。

摘要(英)

Copper is an essential micro-nutrient for normal plant growth and behaves as an un-replaceable enzyme cofactor. Copper, under excess condition in organisms, will enhance the production of reactive oxygen species (ROS) due to Fenton reaction. These ROS will further damage the cells. To avoid this situation, there must be some detoxification mechanisms in plants. So far, the global gene research of the response to excess copper in plants hasn’t been reported. We used the reverse subtracted cDNA microarray, IBS Tomato-Array and CGEP Tom1-Array to survey genes which response to excess copper in tomato roots. We found that 22 non-redundant tentative consensus were down-regulated under copper stress, and 72 ones been up-regulated. According to the nature of these genes, we suggest that copper stress has strong correlation with oxidative stress. These up-regulation genes most are ROS scavengers, such as glutathione S-transferase/peroxidase, ascorbate free radical reductase and cytochrome P450. Moreover, some heat shock proteins were also up-regulated which implicates the elevated tolerance to copper. Tonoplast instrinsic protein delta type and vacuolar H+ ATPase B subunit were down-regulated under excess copper. This indicates the activity in vacuole could altered in response to copper stress.

關鍵字(中)

★ 銅逆境

關鍵字(英)

★ copper stress

論文目次

目錄
中文摘要------------------------------------------------------------------------Ⅰ
英文摘要 ----------------------------------------------------------------------Ⅱ
目錄 ----------------------------------------------------------------------------Ⅲ
『圖』目錄 -------------------------------------------------------------------Ⅴ
『表』目錄--------------------------------------------------------------------Ⅵ
『附錄』目錄-----------------------------------------------------------------Ⅶ
縮寫與全名對照表-----------------------------------------------------------Ⅷ
第一章緒論
一、蕃茄簡介 ----------------------------------------------------------1
二、銅在植物中所扮演的角色 -------------------------------------1
三、銅在植物內的恆定機制-----------------------------------------2
四、文獻中已知受過量銅影響之基因-----------------------------3
五、過量銅離子濃度對植物生理的影響--------------------------5
六、研究動機-----------------------------------------------------------5
第二章材料與方法
第一節實驗材料
一、種子的來源---------------------------------------------------------7
二、蕃茄的種植與過量銅濃度處理---------------------------------7
第二節實驗方法
一、訊息核糖核酸之純化 --------------------------------------------8
二、反向相減式雜交法 -----------------------------------------------9
三、建立反向相減式cDNA基因庫--------------------------------15
四、 cDNA微陣列技術 ----------------------------------------------17
五、基因定序與分析--------------------------------------------------21
六、北方墨點法--------------------------------------------------------21
第三章實驗結果
第一節過量銅反向相減式基因庫微陣列之製作
一、製作過量銅反向相減式基因庫-----------------------------------26
二、製作微陣列晶片-----------------------------------------------------26
第二節受過量銅所影響之基因篩選
一、受過量銅所抑制(down-regulation)之基因----------------------28
1. 在Sub-Array中之分析結果---------------------------------------28
2. 在IBS Tomato-Array中之分析結果-----------------------------30
3. 在CGEP Tom1-Array中之分析結果 ---------------------------30
二、受過量銅所誘導(up-regulation)之基因--------------------------31
1. 在IBS Tomato-Array中之分析結果-----------------------------32
2. 在CGEP Tom1-Array中之分析結果 ---------------------------33
第四章討論-----------------------------------------------------------------35
第五章建議-----------------------------------------------------------------42
參考文獻 ---------------------------------------------------------------------43
圖目錄
圖一、製備蕃茄根部組織反向相減式雜交法之品質控制-------------48
圖二、受過量銅抑制之蕃茄根部細胞的基因片段大小之確定-------49
圖三、北方墨點法分析GADPH基因在蕃茄根部受銅逆境下之表現-----------------------------------------------------------------------------50
圖四、Sub-Array 圖像Data 1-----------------------------------------------51
圖五、Sub-Array 圖像Data 2-----------------------------------------------52
圖六、Sub-Array Data1之內控制基因訊號分佈圖，及所有基因在以內控制基因校正後之四重複平均訊號值---------------------------53
圖七、Sub-Array Data2之內控制基因訊號分佈圖，及所有基因在以內控制基因校正後之四重複平均訊號值---------------------------54
圖八、兩次Sub-Array資料間之相關性分析-----------------------------55
圖九、Sub-Array中，受銅抑制之基因片段大小 ------------------------56
圖十、北方墨點法實驗結果-------------------------------------------------57
圖十一、CGEP Tom1-Array受過量銅處理之基因訊號分佈圖--------58
圖十二、Sub-Array中所得之TC，以其比率之平均值，對應於IBS Tomato-Array及CGEP Tom1-Array的比率平均值----------------59
圖十三、楊明華之過量銅正向相減式基因庫之基因訊號分佈-------60
圖十四、在IBS Tomato-Array及CGEP Tom1-Array共有之基因訊號表現倍率對應圖---------------------------------------------------------61
表目錄
表一、Modefied Hoagland 水耕培養液-----------------------------------62
表二、本研究所選用引子(primer)之序列及其長度---------------------63
表三-1~2、Sub-Array之受過量銅抑制的基因序列比對結果----64~65
表四、IBS Tomato-Array中受過量銅影響之基因分析總表 ---------66
表五-1~2、IBS Tomato-Array中受過量銅抑制基因表現小於0.5之基因---------------------------------------------------------------------67~68
表六、於CGEP Tom1-Array受過量銅抑制而基因表現小於0.5之基因 ------------------------------------------------------------------------69
表七、本論文中所有受抑制(down-regulation)之基因TC統整--------70
表八-1~5、IBS Tomato-Array中受過量銅誘導，表現倍率大於2之基因---------------------------------------------------------------------71~75
表九-1~5、於CGEP Tom1-Array受過量銅誘導，表現倍率大於2之基因------------------------------------------------------------------76~80
表十-1~3、IBS-Array及CGEP Tom1-Array受過量銅誘導兩倍以上之已知功能TC總表-------------------------------------------------81~83
附錄
附錄一、植物調控金屬的恆定機制四個步驟----------------------------84
附錄二、(a)酵母菌中已知之銅運輸機制
(b)目前已知植物內與酵母菌相對應之銅運輸功能表------85
附錄三、製備相減式cDNA雜交法實驗流程-----------------------------86
附錄四、北方墨點法實驗流程 --------------------------------------------87
附錄五、免疫探知作用呈色反應-------------------------------------------87
附錄六-1~5、Sub-Array中所有定序基因之Blast結果總表-------88~92

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指導教授

詹明才、黃雪莉
(Ming-Tsair Chan、Shir-Ly Huang)

審核日期

2003-7-18

推文