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研究生:李思涵
研究生(外文):Shin-Han Lee
論文名稱:苦瓜植物生長素輸出運送蛋白基因之選殖與分析
論文名稱(外文):Cloning and Analysis of the Auxin Efflux Carrier Gene in Bitter Gourd
指導教授:杜宜殷黃鵬林
指導教授(外文):Yi-Yin DoPung-Ling Huang
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:2002
畢業學年度:90
語文別:中文
論文頁數:85
中文關鍵詞:植物生長素輸出運送蛋白基因苦瓜
外文關鍵詞:AuxinAuxin Efflux Carrier GeneBitter Gourd
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以選殖出之苦瓜植物生長素輸出運送蛋白cDNA pMAEC28與pMAEC93為探針,進行溶斑雜交法篩選苦瓜基因組庫,共篩選一百五十萬個溶斑形成單位。依訊號強度差異分等級,挑選40個溶斑進行純化後,共獲得34個選殖系。經由選殖系歸類之雜交結果,將31個選殖系分為二類,得到15個相對應於cDNA pMAEC28的選殖系與16個相對應於pMAEC93的選殖系,選取相對應於pMAEC28之選殖系λMCm-AEC1-34進行核酸定序及分析。
苦瓜植物生長素輸出運送蛋白相關基因MCm-AEC1基因本體全長共有3,206個鹼基對,包含六個顯子及五個隱子,顯子與隱子交界處皆遵守AG-GT原則,與cDNA核酸序列之差異造成演繹蛋白有二個胺基酸不同。與阿拉伯芥( PIN1、PIN2、PIN3、PIN4、PIN6、PIN7 )、芥菜( BjPIN2、BjPIN3 )、水稻( REH1 )植物生長素輸出運送蛋白相關基因胺基酸序列同源性介於45.6~76.61%間,與阿拉伯芥PIN1基因關係最近。在N端第1至26個胺基酸皆存在一可能之signal peptide,並於序列中亦皆可找到數個重複相似之序列。TATA box位於轉譯起始點前449 ~ 455 bp處、CAAT box位於轉譯起始點前570 ~ 574 bp處,並於TATA box、CAAT box上游存在數個可能受植物生長素、乙烯、光、水楊酸、創傷等因子誘導調控基因表現之保守性序列。為進行啟動子活性分析,將啟動子序列構築至載體中,得到質體pMB,此質體中含有苦瓜植物生長素輸出運送蛋白MCm-AEC1基因全長之啟動子序列、GUS報導基因及NOS終結子。
北方雜交分析結果顯示以10-3 M IAA處理0.5及1.25小時、10-3 M IBA處理1.25小時、10-3 M NAA處理1.25小時與10-3 M 2,4-D處理1.25及1.75小時之條件可偵測到基因表現量外,其餘處理條件則只有微量之mRNA累積,無顯著誘導結果可見。
The putative auxin efflux carrier gene MCm-AEC1 from bitter gourd was isolated from a genomic library constructed in the λEMBL3 vector by screening with pMAEC28 and pMAEC93 cDNAs. Approximately 1.5x106 plaque-forming units were screened by plaque hybridization and 34 putative clones were purified individually. According to the results of dot hybridization and restriction endonuclease site map, the auxin efflux carrier genomic clones were classified into two groups. One of the genomic clones corresponding to pMAEC28,λMCm-AEC1-34, was completely sequenced and characterized.
MCm-AEC1 gene in λMCm-AEC1-34, spanning 3,206 base pairs, contains six exons and five introns with consensus AG-GT dinucleotides locating at their boundaries. The amino acid sequences of bitter gourd auxin efflux carrier shows 45.6~76.61% homology to auxin efflux carrier of Arabidopsis, Brassica and rice. The first 26 amino acids are predicted to be a putative signal peptide. Four conserved repeats exist in the polypeptide. The putative TATA and CAAT boxes are 449~455 bp and 570~574 bp upstream from the translation start site in MCm-AEC1, respectively. Several conserved elements responsive to auxin, ethylene, light, salicylic acid and wounding are found in the promoter region. Northern blot analysis indicated that the expression of bitter gourd auxin efflux carrier gene MCm-AEC1 was induced by 10-3 M IAA, IBA, NAA and 2,4-D. No obvious gene expression was detected in other treatments.
To analyze the promoter activity, 3 kb promoter fragment was fused to GUS reporter gene coding sequence for further studies.
英文摘要………………………………………………………………………1
中文摘要………………………………………………………………………2
壹、前言………………………………………………………………………3
貳、前人研究…………………………………………………………………4
一、植物生長素之生理功能…………………………………………………4
二、植物生長素之及極性運輸與結合蛋白…………………………………4
(一)、植物生長素之輸出運送蛋白………………………………………6
(二)、植物生長素之輸入運送蛋白……………………………………..10
(三)、植物生長素之結合蛋白……………………………………………11
三、植物生長素誘導之基因表現……………………………………………14
參、材料與方法………………………………………………………………16
一、植物材料…………………………………………………………………16
二、啟動子片段之合成………………………………………………………16
(一)、GenomeWalker模版DNA之製備.............……………………16
(二)、聚合酶連鎖反應(Polymerase Chain Reaction,PCR)..………16
(三)、大腸桿菌待轉型細胞(competent cells)之製備………..…….17
(四)、質體DNA之轉型…………………………………………………….18
(五)、質體DNA之小量製備……………………………………………….18
(六)、核酸探針之製備……………………………………………………19
(七)、南方氏雜交分析……………………………………………………19
三、苦瓜植物生長素輸出運送蛋白基因之選殖與序列分析………………20
(一)、苦瓜基因組庫之來源………………………………………………20
(二)、苦瓜基因組庫之篩選………………………………………………20
(三)、限制酶圖譜分析……………………………………………………21
(四)、DNA定序...............................................22
四、苦瓜植物生長素輸出運送蛋白基因之表現分析………………………22
(一)、北方雜交分析………………………………………………………22
1. 苦瓜材料之處理………………………………………………………….22
2. 苦瓜RNA之抽取…………………………………………………………..22
3. 北方雜交分析…………………………………………………………….23
五、苦瓜植物生長素輸出運送蛋白基因啟動子之構築……………………23
肆、結果………………………………………………………………………25
一、啟動子片段之合成………………………………………………………25
二、苦瓜植物生長素輸出運送蛋白相關基因之選殖與序列分析…………25
(一)、苦瓜植物生長素輸出運送蛋白相關基因之選殖…………………25
(二)、苦瓜植物生長素輸出運送蛋白相關基因選殖系之限制酶圖譜分析30
(三)、苦瓜植物生長素輸出運送蛋白相關基因之序列分析……………39
三、苦瓜植物生長素輸出運送蛋白相關基因之表現分析…………………48
四、苦瓜植物生長素輸出運送蛋白基因啟動子之構築……………………48
伍、討論………………………………………………………………………………63
一、啟動子片段之合成………………………………………………………63
二、苦瓜植物生長素輸出運送蛋白相關基因之選殖與序列分析…………64
(一)、苦瓜植物生長素輸出運送蛋白相關基因之序列分析……………64
(二)、苦瓜植物生長素輸出運送蛋白相關基因啟動子之序列分析……67
三、苦瓜植物生長素輸出運送蛋白相關基因之表現分析…………………72
陸、結語………………………………………………………………………76
參考文獻………………………………………………………………………77
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