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研究生: 林宛蓁
Lin, Wan-Chen
論文名稱: 轉錄因子調控阿拉伯芥成熟化過程與癒傷組織形成之分析
Molecular studies on the role of transcription factors in plant maturation and callus formation
指導教授: 黃浩仁
Huang, Hao-Jen
學位類別: 碩士
Master
系所名稱: 生物科學與科技學院 - 熱帶植物科學研究所
Institute of Tropical Plant Sciences
論文出版年: 2010
畢業學年度: 98
語文別: 英文
論文頁數: 121
中文關鍵詞: 轉錄因子成熟癒傷組織阿拉伯芥下胚軸
外文關鍵詞: maturation, callus formation, hypocotyl, Arabidopsis thaliana
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  • 阿拉伯芥的生長週期可以區分為幾個階段:種子萌芽、幼苗成長、幼芽成熟及開花進入生殖繁殖階段。而在植物生長的過程中,衰老是一種與年紀相關的計畫性死亡過程,並且會導致植物生長邁向終點。然而在適當的組織培養條件下,已分化的植物細胞可具有去分化及再生新組織的能力。值得注意的是在細胞去分化的過程中,基因表現量的改變扮演重要調控角色。基因的表現量是在轉錄階段受到轉錄因子所調控的,且轉錄因子會在不同的生理反應途徑中參與調控基因表現量。因此,在本研究中將探討轉錄因子與植物生長及癒傷組織形成的相關性。首先,利用比較基因體學方法,分析在阿拉伯芥下胚軸成熟階段與自然凋亡或是受到黑暗誘導之葉片衰老的過程中,試圖找出調控植物成熟相關的轉錄因子。為了更進一步瞭解轉錄因子的功能,挑選三個在誘導癒傷組織形成時基因表現量受到抑制的基因,來進行相關的基因功能性分析。這三個基因分別是AtMBK20.1、AtMBF1c和AtTGA1。利用洋蔥上表皮短暫表現GFP融合蛋白實驗證實,轉錄因子AtMBK20.1和 AtTGA1表現位於細胞核內。然而在大量表現AtMBK20.1、AtMBF1c或AtTGA1的阿拉伯芥下胚軸其癒傷組織的形成則是和對照組相同的,並沒有因為基因的大量表現而有受到抑制的現象。因此,再利用比較基因體學方式,將誘導癒傷組織生長的過程與幼芽生長時受到的調控的轉錄因子進行相互比較,發現有五個轉錄因子的基因調控型態是相同的,分別是MYC2、MYBC1、F25A4.19、F6N18.8 和 GT-2,顯示這些轉錄因子可能參與細胞去分化及再生的過程。上述所得結果有助於我們更加瞭解哪些植物轉錄因子會參與調控植物成熟與誘導癒傷組織的形成。

    The life cycle of Arabidopsis thaliana can be divided into several developmental phases,form seed germination, seedling growth, shoot maturation and the transition to flowering.Senescence is the process of age-dependant programmed cell death leading to end of a lifespan. However, plant cells have the capability to reverse their state of differentiation and regenerate new tissues under cultured condition. Cellular dedifferentiation is characterized by remarkable changes in the pattern gene expression. Gene expression is regulated predominantly at the transcriptional level, and numerous transcription factors act key regulator of various biological process. In this study, attempts were made to understand the involvement of maturation-related or callus formation-associated transcription factors. A comparative transcriptome analysis for successive stages of A. thaliana developmental hypocotyl maturation, natural leaf senescence and dark-induced leaf senescence uncovered novel maturation-associated transcriptional networks with distinct expression profiles. To gain more insight into the functional significance of the transcription factors, transgenic 35S-MBK20.1, 35S-MBF1c and 35S-TGA1 Arabidopsis plants were generated. However, the capacity for callus formation was unchanged and similar to that of empty vector-transformed plants. Transient expression analysis in onion epidermal cells indicated the nuclear localization for the AtMBK20.1-GFP and AtTGA1-GFP fusion proteins. A comparison of gene expression in callus formation and shoot apex growth revealed five transcription factors, which may be associated in cell dedifferentiation and organ regeneration. In summary, the study identified the maturationor dedifferentiation-associated transcription factor genes. These results will be helpful with isolation of transcription factors involved in age-dependant callus formation.

    Content 致謝 ……………………………………………………….I 中文摘要 ............................................ II Abstract ............................... III Content .................................. IV Figure content ..................................... VI Supplement table content ........................... VIII Abbreviations .............................. X 1.Introduction ................................... 1 1.1 Plant growth and development..................... 1 1.2 Leaf senescence ........................... 2 1.3 Development of hypocotyls ..................... 3 1.4 Transcription factors ........................ 3 1.5 Dedifferentiation in animals ...................... 6 1.6 Dedifferentiation in plants .................... 6 1.7 Auxin and cytokinins ......................... 7 1.8 Cell-cycle and cell division ................... 8 1.9 Aims of this study ........................... 10 2. Materials and methods .......................... 11 2.1 Microarray data analysis ........................ 11 2.2 Plant materials and growth conditions ............. 11 2.3 Tissue culture ................................ 12 2.4 Preparation of total RNA ....................... 12 2.5 Total RNA clean-up ............................ 13 2.6 Semi-quantitative reverse transcription PCR (RT-PCR) .............................. 13 2.7 Tissue-specific expression of AtMBK20.1, AtTGA1 and AtMBF1c .............. 14 2.8 Subcellular localization of AtMBK20.1 and AtTGA1 proteins .................... 14 2.9 AtMBK20.1, AtMBF1c and AtTGA1 fragments production by PCR ............. 15 2.10 Purification of PCR product .................. 16 2.11 Plasmid construction ......................... 17 2.12 Plant transformation ......................... 18 2.13 Identification of transgenic lines in Arabidopsis and examination of endogenous AtMBK20.1, AtMBF1c and AtTGA1 transcript in the transgenic lines........ 18 2.14 Callus induction .............................. 19 2.15 Histological observations ..................... 19 2.16 Salt and heat stress analysis ................. 19 3.Results ....................................... 21 3.1 Transcription factors genes up- or down-regulated during the hypocotyl maturation in Arabidopsis ......................... 21 3.2 Comparison of expression profiles for transcription factor genes between hypocotyls maturation and natural leaf senescence in Arabidopsis ..................... 21 3.3 Comparison of expression profiles for transcription factor genes between hypocotyls maturation and dark-induced leaves senescence in Arabidopsis ....... 22 3.4 Transcription factors up- or down-regulated during callus formation ........... 23 3.5 Selection of candidate genes involved in the callus formation from microarray data and srd2 temperature sensitive mutant confirmation by semi-quantitative RT-PCR ........................... 24 3.6 Tissue specific expression of AtMBK20.1, AtMBF1c and AtTGA1 ............... 25 3.7 AtMBK20.1 and AtTGA1 are nucleus localized proteins ............................. 25 3.8 Analysis of gene expression in transgenic 35S-MBK20.1, 35S-MBF1c and 35S-TGA1 Arabidopsis plants ................................... 26 3.9 Transgenic plants of callus formation ........................................ 26 3.10 Comparison of expression profiles for transcription factor genes between callus formation from root and hypocotyl explants ............................................. 27 3.11 Comparison of expression profiles for transcription factor genes between hypocotyls-derived callus formation and T87 cell versus root explants .............. 28 3.12 Comparison of expression profiles for transcription factor genes between callus formation and shoot apex growth ....................................... 28 3.13 The effect of salt stress on 35S-TGA1 transgenic Arabidopsis plants ......... 29 3.14 The effect of heat stress on 35S-TGA1 transgenic Arabidopsis plants ........ 29 4.Discussion ........................................ 30 4.1 Transcriptional regulation during hypocotyls maturation .............................. 30 4.2 Transcriptional regulation during callus formation ........................................ 31 4.3 AtMBK20.1 and AtTGA1 were highly expressed in mature tissue and localized in nuclei ........................................ 32 4.4 Callus formation was not affected in transgenic 35S-MBK20.1, 35S-MBF1c and 35S-TGA1 Arabidopsis plants ............................................ 33 4.5 35S-TGA1 transgenic plants in abiotic stress condition ................................. 33 5. Future work .................................... 35 References ......................................... 36 Figure content Figure 1. Families of transcription factors showing alteration in expression in mature Arabidopsis hypocotyls……………………………………………………45 Figure 2. Comparison of expression profiles for transcription factor genes between hypocotyl maturation and leaf senescence………………………………...46 Figure 3. Venn diagrams showing the numbers of significantly up- (A) or down-regulated (B) transcription factor genes that are unique or commonly regulated during CIM incubation in juvenile or mature stage…………….47 Figure 4. The significantly up- or down-regulated genes in transcription factor families that unique or commonly regulated during CIM incubation in juvenile or mature stage…………………………………………………...48 Figure 5. Gene expression of AtMBK20.1, AtMBF1c and AtTGA1 transcription factors in Arabidopsis hypocotyls during CIM incubation……………………….50 Figure 6. RT-PCR analysis of AtMBK20.1, AtTGA1 and AtMBF1c transcripts in various organs……………………………………..………………………51 Figure 7. Nuclear localization of the AtMBK20.1-GFP and AtTGA1-GFP proteins..52 Figure 8. A map of three constructs used in the transformation of Arabidopsis, and a construct encoding GFP was used as an empty vector (EV) control……...53 Figure 9. The expression levels of AtMBK20.1, AtTGA1 and AtMBF1c in transgenic Arabidopsis plants. ……………………………………………………….54 Figure 10. Gene expression of AtMBK20.1 in Arabidopsis hypocotyls during CIM incubation…………………………………………………………………55 Figure 11. Callus formation from hypocotyls explants in transgenic 35S-MBK20.1 and empty vector control…………………………………………………...….56 Figure 12. Callus formation from hypocotyls explants in transgenic 35S-TGA1 empty vector control. ……………………………………………………………..57 Figure 13. Callus formation from hypocotyls explants in transgenic 35S-MBF1c and empty vector control………………………………………………………58 Figure 14. Venn diagrams showing the numbers of significantly up- (A) or down-regulated (B)t transcription factor genes that unique or commonly regulated during callus formation form root, juvenile hypocotyl and mature hypocotyl calli……………………………………………………………..59 Figure 15. Venn diagrams showing the numbers of significantly up-regulated (A) and down-regulated (B) transcription factor genes that unique or commonly regulated in T87 calli vs. freshly-derived calli (Habituation), juvenils hypocotyl calli and mature hypocotyl calli……………………………….60 Figure 16. Venn diagrams showing the numbers of significantly up-regulated (A) and down-regulated (B) transcription factor genes that unique or commonly regulated in shoot apex growth, juvenils hypocotyl calli and mature hypocotyl calli……………………………………………………………..61 Figure 17. Effect of salt stress on 35S-TGA1 transgenic Arabidopsis plants………...62 Figure 18. Effect of heat stress on 35S-TGA1 transgenic Arabidopsis plants…….….63

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