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Student Number 942204007
Author Ling-Jen Ma(馬翎甄)
Author's Email Address No Public.
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Department Life Science
Year 2006
Semester 2
Degree Master
Type of Document Master's Thesis
Language zh-TW.Big5 Chinese
Title Functional Analysis of AtMYBS Genes in Arabidopsis
Date of Defense 2006-07-18
Page Count 72
Keyword
  • arabidopsis
  • MYB
  • signal transduction
  • sugar
  • Abstract MYB proteins are transcription factors that contain conserved DNA binding domains, and comprise a large gene family which can be grouped into several subfamilies by phylogenic criteria. In plants, there are three types of MYB proteins, R1R2R3, R2R3, and R1/2. Previously, we identified three novel R1/2 type MYB proteins in rice, OsMYBS1, OsMYBS2, and OsMYBS3. They contain a single repeat of MYB DNA binding domain, and are involved in sugar- and hormone-regulated α-amylase genes expression mechanism. On the other hand, similar proteins in barley, HvMCB1 and HvMYBS3 have been reported that they are important transcription factors during barley seed development. However, the physiological functions of MYBSs in plants are still not well known. As Arabidopsis is a powerful model plant, supported by robust bioinformatics and comprehensive mutant collection, we use Arabidopsis to further investigate biological roles of MYBSs in plants.
    In this study, four different full-length AtMYBS cDNAs were isolated from Arabidopsis, and these genes were expressed ubiquitously in roots, leaves, flowers and siliques with their own unique expression pattern. To study physiological roles of these genes, a gain-of-function strategy was applied by generating over-expression of AtMYBSs transgenic Arabidopsis plants; meanwhile, loss-of-function method was used by screening T-DNA insertion mutants. MYBSs over-expression transgenic lines and T-DNA mutant lines has been identified in this work. These plant materials will be valuable for function analysis on AtMYBSs in the near future. The other part of this study was found gene expressions of AtMYBSs are sugar respondent. Like MYBSs in rice and barley, the TA-box of sugar response element is specifically recognized by AtMYBSs, implying AtMYBSs may play roles in regulation of plant sugar metabolism.
    Table of Content 英文摘要…………………………………………………………….......i
    中文摘要………………………………………………………………...ii
    誌謝………………………………………………………………...iii
    目錄………………………………………………………………...iv
    圖目錄………………………………………………………………...viii
    表目錄………………………………………………………………...x
    縮寫檢索表………………………………………………………………...xi
    壹、緒論
    第一部分、植物糖訊息傳遞及基因調控的研究............................................1
    1. 酵母菌中糖的訊息傳遞.........................................................................1
    2. 植物對糖的感應及訊息傳導.................................................................2
    2.1.糖在植物中扮演訊息傳遞的角色...........................................2
    2.2.植物對糖的感應及訊息傳導...................................................3
    第二部分、水稻中糖訊息傳導途徑................................................................5
    1. MYB家族的結構特性與分類.................................................................5
    2. 水稻萌芽時期α-澱粉水解酵素之活化調控機轉................................6
    3. α-澱粉水解酵素啟動子TA-box與轉錄活化子MYBSs作用機制.....7
    第三部分、阿拉伯芥 (Arabidopsis) 簡介......................................................8
    1. 雙子葉模式植物.....................................................................................8
    2. Arabidopsis R1/2 MYB 基因..................................................................9
    貳、材料與方法
    第一部分、菌株、載體與植物品系..................................................................10
    第二部分、質體的構築....................................................................................10
    1. 反轉錄PCR合成AtMYBSs基因全長DNA片段...................................10
    1.1植物細胞total RNA的抽取.....................................................10
    1.2RNA的純化 (去除DNA)........................................................11
    1.3反轉錄.......................................................................................11
    1.4設計引子...................................................................................12
    1.5以PCR合成DNA片段............................................................12
    2. 接合反應.................................................................................................13
    2.1限制酵素作用...........................................................................13
    2.2瓊脂膠體回收DNA.................................................................13
    2.3齊頭端DNA片段的接合.........................................................14
    3.細菌的轉殖作用.......................................................................................14
    3.1製備E.coli 勝任細胞 (JM109 strain).....................................14
    3.2細菌的轉殖...............................................................................15
    3.3小量純化細菌plasmid DNA法...............................................15
    3.4大量純化細菌plasmid DNA法...............................................16
    4. DNA序列分析.........................................................................................17
    第三部份、轉植株基因型的分析....................................................................17
    1. T-DNA插入突變之轉殖植株獲得..........................................................17
    1.1訂購轉殖株...............................................................................17
    1.2種植條件...................................................................................17
    2. 轉殖植株基因型鑑定.............................................................................17
    2.1抽取genomic DNA...................................................................17
    2.2PCR確認植株的基因型..........................................................18
    2.3RT-PCR確認植株的表現型.....................................................18
    第四部份、農桿菌之阿拉伯芥基因轉殖........................................................19
    1.農桿菌的轉殖...........................................................................................19
    1.1製備Agrobacterium 勝任細胞 (EHA105 strain)....................19
    1.2農桿菌的轉殖方法...................................................................19
    1.3農桿菌生化檢測 (Ketolactose test)........................................19
    2.阿拉伯芥的轉殖.......................................................................................19
    第五部份、轉殖株的分析................................................................................20
    1. GUS染色分析..........................................................................................20
    2. 轉殖株之基因型分析.............................................................................20
    第六部份、Oryza sativa短暫表現分析............................................................20
    1. 基因槍轉殖方式.....................................................................................20
    1.1水稻胚材料之處理...................................................................20
    1.2金粒子的製備...........................................................................21
    1.3DNA coating.............................................................................21
    1.4基因槍類型及型號...................................................................21
    2. 蛋白質的純化與分析.............................................................................22
    2.1收集水稻胚癒傷組織表現之蛋白質.......................................22
    2.2Firefly Luciferase酵素活性分析.............................................22
    2.3GUS酵素活性分析..................................................................22
    叁、實驗結果
    1. AtMYBSs基因的選殖...............................................................................23
    1.1資料庫比對分析.......................................................................23
    1.2選殖AtMYBSs基因..................................................................24
    2. AtMYBSs對TATCCA序列結合專一性分析.........................................24
    3. 分析阿拉伯芥中AtMYBSs基因的表現.................................................25
    3.1AtMYBSs基因在阿拉伯芥中的表現模式...............................25
    3.2AtMYBSs基因在含糖/缺糖環境下表現情形................26
    4. 過量表達AtMYBSs之轉殖阿拉伯芥..................................26
    4.1載體之構築與阿拉伯芥AtMYBSs過量表達轉殖株的建立.26
    4.2利用HygR與GUS活性分析轉殖成功之T1阿拉伯芥...........27
    4.3轉殖植株基因型分析...............................................................27
    5. 阿拉伯芥AtMYBSs突變株後代基因型的分析.....................................28
    5.1選購AtMYBSs T-DNA插入突變的轉殖株.............................28
    5.2AtMYBSs T-DNA插入突變轉殖株基因型分析......................28
    5.3觀察AtMYBSs缺失後的外表型..............................................29
    肆、討論
    1. AtMYBSs在阿拉伯芥各組織中基因的表現狀況...................................54
    2. AtMYBSs基因在有糖與缺糖環境下的表現狀況...................................54
    3. AtMYBSs過量表達之轉殖阿拉伯芥的建立與分析.............................55
    4. AtMYBSs基因T-DNA插入突變之轉殖阿拉伯芥分析..........................55
    5. 阿拉伯芥MYBSs基因可能的角色.........................................................56
    5.1.阿拉伯芥MYBSs基因可能參與糖調控基因的表現..............56
    5.2.阿拉伯芥MYBSs基因可能參與光對I-box之基因調控機制57
    參考文獻...................................................................................................58
    附錄一引子列表...................................................................................61
    附錄二質體建構圖A...........................................................................64
    附錄三質體建構圖B...........................................................................65
    附錄四試劑配方...................................................................................67
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    Advisor
  • Chung-An Lu(陸重安)
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    Date of Submission 2007-07-24

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