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研究生:蔡嘉慧
研究生(外文):Tsai, Chia-Hui
論文名稱:香蕉ABC轉運蛋白基因之分析
論文名稱(外文):Analysis of the ABC (ATP-Binding Cassette) Transporter Gene in Banana
指導教授:黃鵬林杜宜殷
指導教授(外文):Huang, Pung-LingDo, Yi-Yin
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:園藝學研究所
學門:農業科學學門
學類:園藝學類
論文種類:學術論文
論文出版年:2001
畢業學年度:89
語文別:中文
論文頁數:79
中文關鍵詞:香蕉ABC 轉運蛋白
外文關鍵詞:BananaATP-Binding Cassette transporter
相關次數:
  • 被引用被引用:9
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將以香蕉ABC轉運蛋白(ATP binding cassette transporter)同源cDNA選殖系pBER44 cDNA為探針,篩選得到之基因組選殖系lABC13、14、18、98-1、123,進行限制酶圖譜分析及基因組DNA南方氏雜交分析,得知lABC18、98-1及lABC123選殖系均包含完整的pBER44基因片段,其中lABC123具較長的5’端。選取lABC18、123基因組選殖系,進行核酸定序分析,得到香蕉ABC轉運蛋白基因及啟動子序列,顯示此基因至少具有24個顯子及23個隱子,顯子與隱子連接部分的核苷酸序列皆遵循GT-AG規則。此基因解碼的蛋白具有1,425個胺基酸,分子量為161 kDa,等電點為7.09;一級結構方面,以ATP結合區域和跨膜組區為一單元,重複出現一次,其中每個跨膜組區各有6個α-helix結構,其蛋白質結構與酵母菌ABC轉運蛋白PDR5基因家族相同。經比對香蕉ABC轉運蛋白與水稻、阿拉伯芥、煙草、浮萍之ABC轉運蛋白胺基酸序列,相似度均在69 %以上。分析其啟動子序列,推測在轉譯起始點上游1263-1269 bp處為TATA box,這個區域可能含有一個顯子及隱子。在TATA box上游也存在數個可能受到gibberellin A3、abscisic acid及低溫調控之保守性序列。
由北方雜交分析結果顯示,香蕉ABC轉運蛋白基因在遭受缺水逆境的葉片內有明顯的表現量,在苞片、雄蕊、雌蕊、子房及懸浮細胞內則mRNA累積偏低;果實後熟過程中成熟度七級的果肉及成熟度五級的果皮中,可偵測到mRNA有顯著的累積。隨著成熟度的增加,低溫處理可抑制香蕉ABC轉運蛋白基因之表現;以不同濃度ABA處理成熟度一級果實切片12小時,基因表現量亦不高。以不同誘導物處理成熟度五級之果實切片及果皮,以10-3M 6-benzyl-adenine、10-3M ABA、10-1M NaCl處理,在切片及果皮中均可微量提高mRNA累積量,而以10-3M IAA處理則會減少基因的表現量。
Banana genomic clones λABC13, 14, 18, 98-1 and 123 isolated by using banana ABC transporter homology cDNA clone pBER44 as probe, can be classified into two groups based on restriction site maps and Southern analysis. All three clones λABC18, 98-1, 123 contain the full-length of pBER44 cDNA. λABC123 possesses more 5’ upstream sequence than λABC18 and 98-1. The banana ABC transporter gene consists of at least 24 exons and 23 introns with consensus GT-AG dinucleotides locating at their boundaries. The putative banana ABC transporter consists of 1,425 amino acids with a calculated molecular mass of 161 kDa and a predicted isoelectric point of 7.09. It contains two copies each of two structural units, a peripherally located ATP binding domain and a highly hydrophobic transmembrane domain with six transmembrane-spanning α-helices, and displays similarity to the typical structure and domain organization of the yeast PDR5 gene family. The amino acid sequence of banana ABC transporter shows at least 69 % homology to ABC transporter from Arabidopsis, rice, Nicotiana plumbaginifolia (NpABC1) and Spirodela polyrrhiza (TUR2). According to the result of promoter sequence analysis, a putative TATA box is 1263-1269 bp upstream the translation start site. Several consersved elements responsive to ABA, GA and low temperature are found in the promoter region. Northern blot analysis indicates that banana ABC transporter gene expression was induced by drought in leaves, and the mRNA accumulated abundantly in the pulp at ripening stage 7 and peel at ripening stage 5. Repression of banana ABC transporter gene expression by low temperature is consistent with fruit ripening. No obvious gene expression was detected in banana fruit at ripening stage 1 by different concentrations of ABA treatment for 12 hours. Accumulation of banana ABC transporter mRNA was enhanced in fruits at ripening stage 5 treated with 10-3M BA, 10-3M ABA or 10-1M NaCl for 12 hours. However, its expression was reduced by 10-3M IAA treatment.
中文摘要…………………………………………………………………….…1
英文摘要……………………………………………………………………….2
壹、前言……………………………………………….…..4
貳、前人研究………………………………………………………...5
(一)ABC轉運蛋白的結構……………………………………………....5
(二)MDR(multidrug resistance)family………………..……...6
(三)MRP(multidrug resistance associated protein)subfamily..7
(四)PDR5(pleiotropic drug resistance)subfamily……………..9
參、材料與方法……………………………………………………..11
一、試驗材料………………………………………………….…..11
(一)植物材料……………………………………………………….…...11
(二)香蕉ABC轉運蛋白基因組選殖系之來源…………………………11
二、試驗方法…………………………………………….………..12
(一)南方氏雜交分析………………………………………….………...12
1. 核苷酸探針之製備……………………………………………………12
2. 南方氏雜交分析………………………………………………………13
(二)限制酶圖譜分析…………………………………………………….14
(三)次選殖……………………………………………………………....14
1. 大腸桿菌待轉型細胞(competent cells)之製備……………………14
2. 接合反應(ligation)與質體DNA之轉型…………………………….15
3. 質體之小量製備……………………………………………………....15
4. DNA定序…………………………………...…………………………16
(四)北方雜交分析……………………………………………………….16
1. 香蕉材料之處理……………………………………………………..16
(1)植株不同部位之基因表現…………………………………..…16
(2)不同成熟度果實之基因表現……………………………………16
(3)果實於不同溫度下之基因表現…………………………………17
(4)果實於不同ABA(abscisic acid)濃度處理下之基因表現……17
(5)果實於不同誘導物處理下之基因表現…………………………18
2. 香蕉RNA之抽取…………………………………………………….18
3. 北方雜交分析………………………………………………………..19
肆、結果………………………………………………………….20
一、香蕉ABC轉運蛋白基因結構分析………………………………20
(一)香蕉ABC轉運蛋白限制酶圖譜分析………………………………20
(二)香蕉ABC轉運蛋白基因組DNA南方式雜交分析………………20
(三)香蕉ABC轉運蛋白基因組DNA序列之分析……………………27
二、香蕉ABC轉運蛋白基因表現分析……………………………49
(一)植株不同部位之基因表現…………………………………………49
(二)不同成熟度果實之基因表現………………………………………49
(三)果實於不同溫度下之基因表現……………………………………51
(四)果實於不同ABA濃度處理下之基因表現…………………………58
(五)果實於不同誘導物處理下之基因表現……………………………58
伍、討論……………………………………………………………....63
一、香蕉ABC轉運蛋白之序列分析………………………………….63
(一)核苷酸及胺基酸序列之分析……………………………………....63
(二)基因啟動子序列之分析…………………………………………….68
二、香蕉ABC轉運蛋白之基因表現………………………………..70
(一)香蕉ABC轉運蛋白基因於香蕉植株不同部位之表現……………70
(二)香蕉ABC轉運蛋白基因於不同成熟度果實內的表現……………71
(三)香蕉ABC轉運蛋白在不同處理下之果實內的表現………………72
陸、參考文獻……………………………………………………………..…75
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