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研究生:阮建達
研究生(外文):Jian-Da Juan
論文名稱:分析水稻CysteineProteinase基因(OsEP3A)啟動子受氮素調控之反應序列
論文名稱(外文):Characterization of the Nitrogen Response Sequence in a Rice Cysteine Proteinase Gene Promoter, OsEP3A
指導教授:侯新龍
指導教授(外文):Shin-Lon Ho
學位類別:碩士
校院名稱:國立嘉義大學
系所名稱:農業生物技術研究所
學門:農業科學學門
學類:農業技術學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:70
中文關鍵詞:啟動子蛋白酶
外文關鍵詞:promoterproteinase
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已知水稻OsEP3A ( Oryza sativa endoproteinase 3A )基因啟
動子活性受到缺氮所專一性誘導表現。本實驗為進一步瞭解其受氮素
調控的序列,將OsEP3A 5’端的啟動子縮減成不同長度的片段,分別
利用原生質體短暫性表現分析及利用農桿菌轉殖作穩定性分析。將7
個不同長度的構築載體(construct)轉殖到水稻癒傷組織中,並利用
PCR 方式將轉型細胞的標記基因Hph 擴增出來,確定我們的轉型細胞
系擁有外源基因,共獲得105 個轉型細胞系。將轉型的細胞培養為穩
定之懸浮細胞,再經由含氮(+N)及缺氮(-N)處理後,測量報導基因
Luciferase 的活性表現。發現全長啟動子(EP837)、及啟動子縮短至
EP542 及EP361 時,缺氮處理之Lucierase 活性表現均大於含氮;而
縮短到EP236 以下(包含EP183 及EP113),Luciferase 活性幾乎不表
現,推測受氮素調控的區域在-236 ~ -361 之間。另外,也將實驗室
現有的EPpromoter:GUS 表現載體的T1 種子誘導為癒傷組織,並培
養為懸浮細胞,經缺氮處理後抽取RNA 做北方墨漬雜合分析,其表現
載體啟動子片段分別為EP9(-390);EP4(-278);EP3(-204);
EP5(-144);EP20(-109)及EP19(-60)。EP4 及EP9 仍受缺氮所誘導表
現;而EP3、EP5、EP19 及EP20 則不受缺氮誘導表現,推論調控區為
-204 ~-278 之間。與本實驗Luciferase 活性分析結果所得調控區域
-236 ~ -361 之間有重疊性,因此以夾擊的方式推論氮素調控區域可
能為-236 ~ -278之間。並與其他受缺氮誘導表現的CysP基因oryzain
α、β及γ啟動子序列比對分析,發現在相似位置都有相類似的保守
序列RDAAAAYYRCA,推測可能為受氮素調控的序列。
另一實驗目的為希望建立ㄧ低CysP表現及高Cystatin表現量的
水稻細胞系作為外源蛋白表現的母本,利用α-amylase 啟動子來調
控Cystatin 的表現,我們利用農桿菌基因轉殖的方式成功獲得了12
個品系的轉殖水稻,待T1 種子收成後可做進一步的分析。
It has been known that the rice cysteine proteinase (OsEP3A) gene expression was up-regulated by nitrogen starvation and down-regulated while cells were supplied with nitrogen nutrients. The purpose of this study is to identify the nitrogen response sequence of OsEP3A promoter,
we expressing the luciferase reporter gene driven by the differentially length of 7 of OsEP3A promoters in transformed rice cells. PCR to amplify the antibiotic selectable marker gene, Hph, has provided the evidence that those antibiotic resistance of calli were transformed cell
lines. The transformed calli were cultured as suspension cells, and then were cultured in media with or without nitrogen for the luciferase activity assay. After analyzes the effects of nitrogen on the transcriptional activity
of those various length of OsEP3A promoter, we showed that the nitrogen response sequence may be located on -236 – -361 regions. Beside, we were also induced the calli as suspension cultured cells from the available T1 seeds of EP promoter:GUS transgenic rice, and were cultured in nitrogen starvation medium for 8 days, then purified the total RNA for Northern blot assay. There were six of various expression vector tested: EP390,EP278,EP204,EP144,EP109, and EP60. The GUS reporter gene expression from the EP278 and EP390 transgenic plants were still inducible by nitrogen starvation, and others did not. These result
indicated the nitrogen regulate region is located at -204 to -278. Comparison with the result from the luciferase activity assay, the overlap sequence is from -236 to -278. Further more, we blast the other CysP gene promoter sequences, oryzain α、β and γ,, their genes expression were
also induced by nitrogen starvation, and predicted concensus nitrogen response sequence is : RDAAAAYYRCA .
Another study is to set up a rice cell line, which is coordinately regulated genes expression with a lower and higher manners of CysP and Cystatin, respectively. We have introduced the Cystatin gene into rice plant under the control of α-amylase promoter by Agrobacterium-mediated gene transformation. There were 12 of independent of transgenic rice plants growing in pods, and should be analyze after the T1 seeds have harvested.
目錄 …………………………………………………. Ⅰ
圖目錄 …………………………………………………. Ⅲ
表目錄 ………………………………………………… Ⅳ
摘要 …………………………………………………. Ⅴ
Abstract …………………………………………………. Ⅵ
第一節 前人研究………………………………………. 1
第二節 材料與方法……………………………………. 10
第三節 結果…………………………………………… 24
ㄧ、水稻OsEP3A啟動子受氮素調控的序列分析。………… 24
(1) 水稻OsEP3A啟動子的擴增及表現載體之構築。……… 25
(2) 不同長度OsEP3A啟動子在水稻原生質體中的短暫性表現。25
(3) 構築農桿菌之雙偶型載體(binary vector)。………… 26
(4) 水稻轉型細胞系(transformed cell line)之篩選。… 26
(5) 篩選成功轉型細胞系及誘導轉殖株再生。……………… 26
(6) 轉型細胞系Luciferase 活性分析。……………………… 27
二、轉殖水稻CysP inhibitor, cystatin I,抑制cystein proteinase 的表現分析。……………………………… 29
第四節 討論…………………………………………… 31
參考文獻 ………………………………………………… 55
附錄一 …………………………………………………. 59
附錄二 …………………………………………………. 63
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