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姓名 馬翎甄(Ling-Jen Ma) 查詢紙本館藏 畢業系所 生命科學系 論文名稱 阿拉伯芥 AtMYBS 基因功能性探討
(Functional Analysis of AtMYBS Genes in Arabidopsis)相關論文 檔案 [Endnote RIS 格式]
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至系統瀏覽論文 ( 永不開放)
摘要(中) MYB家族是一群能夠結合特定DNA序列的轉錄因子。在植物中，依照MYB的DNA結合區域 (Binding motif) 數目，將MYB家族分成三大次家族，分別為含有三個DNA結合區域的R1R2R3、兩個DNA結合區域的R2R3及一個DNA結合區域的R1/2次家族。在先前的水稻研究中，發現三個R1/2 MYB蛋白 (OsMYBSs) 參與了糖與賀爾蒙GA調控水稻α-amylase基因的表現。後來在大麥中，相似的R1/2 MYB轉錄蛋白HvMCB1及HvMYBS3也先後被鑑定出參與了種子萌發時期基因表現的調控機轉。然而，截至目前為止，MYBSs在植物體中的生理功能仍然不甚了解，因此在本研究中，選擇以阿拉伯芥為材料，希望針對MYBS做一連串生理功能的探討。
在本論文中，我們分離出四個阿拉伯芥的MYBSs基因，分別命名為AtMYBS1-1、AtMYBS1-2、 AtMYBS2-1與AtMYBS2-3，經由RT-PCR的分析，發現它們幾乎在各個組織器官中都有表現，雖然表現的狀況不盡相同。為了進一步研究MYBSs基因的生理功能，我們分別對阿拉伯芥進行gain-of-function及loss-of-function的研究。我們構築了完整長度的cDNA，利用農桿菌轉殖的方式，建立能夠大量表現MYBSs的阿拉伯芥轉殖株。另一方面，從SALK institute訂購MYBSs基因被T-DNA插入的突變種子，然後篩選得到同型合子的突變植株，這些大量表現與突變植株將有助於往後的研究。
此外，從本研究中，利用RT-PCR的方式，確認AtMYBSs基因的表現是受糖調控的。同時AtMYBSs，如同水稻與大麥中的MYBSs一樣，皆能夠辨認水稻αAmy3啟動子中糖調控序列上的TA-box，顯示了AtMYBSs基因可能參與了植物中糖代謝的調控機轉。摘要(英) 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.關鍵字(中) ★ 糖
★ MYB
★ 阿拉伯芥
★ 訊息傳遞關鍵字(英) ★ arabidopsis
★ signal transduction
★ sugar
★ MYB論文目次 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
1.2 RNA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
2.2 PCR確認植株的基因型.......................................................... 18
2.3 RT-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
1.3 DNA 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
2.2 Firefly Luciferase酵素活性分析............................................. 22
2.3 GUS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
3.1 AtMYBSs基因在阿拉伯芥中的表現模式............................... 25
3.2 AtMYBSs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
5.2 AtMYBSs 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................................................................................... 67參考文獻 Baranowskij, N., C. Frohberg, et al. (1994). A novel DNA binding protein with homology to Myb oncoproteins containing only one repeat can function as a transcriptional activator. Embo J. 13(22): 5383-92.
Chen, P. W., C. M. Chiang, et al. (2006). Interaction between Rice MYBGA and the Gibberellin Response Element Controls Tissue-Specific Sugar Sensitivity of {alpha}-Amylase Genes. Plant Cell 18(9): 2326-40.
Feldbrugge, M., M. Sprenger, et al. (1997). PcMYB1, a novel plant protein containing a DNA-binding domain with one MYB repeat, interacts in vivo with a light-regulatory promoter unit. Plant J 11(5): 1079-93.
Geisler, M., L. A. Kleczkowski, et al. (2006). A universal algorithm for genome-wide in silico identification of biologically significant gene promoter putative cis-regulatory-elements; identification of new elements for reactive oxygen species and sucrose signaling in Arabidopsis. Blackwell Synergy. 45: 384-98.
Jang, J. C., P. Leon, et al. (1997). Hexokinase as a sugar sensor in higher plants. Plant Cell. 9(1): 5-19.
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Rubio-Somoza, I., M. Martinez, et al. (2006). HvMCB1, a R1MYB transcription factor from barley with antagonistic regulatory functions during seed development and germination. Plant J. 45(1): 17-30.
Rubio-Somoza, I., M. Martinez, et al. (2006). Ternary complex formation between HvMYBS3 and other factors involved in transcriptional control in barley seeds. Plant J. 47(2): 269-81.
Schaffer, R., N. Ramsay, et al. (1998). The late elongated hypocotyl Mutation of Arabidopsis Disrupts Circadian Rhythms and the Photoperiodic Control of Flowering. Cell 93(7): 1219-1229.
Sheen, J. (1990). Metabolic Repression of Transcription in Higher Plants. Am Soc Plant Biol. 2: 1027-1038.
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Wang, H. J., A. R. Wan, et al. (2007). Transcriptomic adaptations in rice suspension cells under sucrose starvation. Springer. 63: 441-463.
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Yanhui, C., Y. Xiaoyuan, et al. (2006). The MYB Transcription Factor Superfamily of Arabidopsis: Expression Analysis and Phylogenetic Comparison with the Rice MYB Family, Springer. 60: 107-124.
Yu, J., Hu, S., Wang, J., Wong, G. K. -S., Li, S., Liu, B., Deng, Y., Dai, L., Zhou, Y., Zhang, X., et al. (2002). A draft sequence of the rice genome (Oryza sativa L. ssp. Indica). Science, 296: 79-92.
Zhi-Yong Wang, David Kenigsbuch, Lin Sun, Eitan Harel, May S. Ong, and Elaine M. Tobin. (1997). A Myb-Related Transcription Factor 1s lnvolved in the Phytochrome Regulation of an Arabidopsis Lhcb Gene. Plant Cell, 9: 491-507.指導教授 陸重安(Chung-An Lu) 審核日期 2007-7-24 推文 plurk
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