葉綠體是一重要的植物細胞器。除在光合作用上扮演重要角色外，葉綠體同時也與其他生理過程有關，例如油脂，氨基酸及植物賀爾蒙的生成等等。葉綠體的發育及作用由一系列的調控因子所調控。如有任何一個調控因子無法正常運作，都可能導致葉綠體什至整棵植物無法正常生長及發育。本人在碩士論文研究期間利用T-DNA激活標籤法進行了一項的bzr1-1D抑制子的突變體篩選實驗。在篩選過程中，意外發現一個幼苗階段致死的隱性白化突變體，命名為bt20。bzr1-1D是一個對油菜素内酯（brassinosteroids, BRs）高度敏感的單點突變體，而該單點突變發生於BR信號傳導中的轉錄因子BZR1基因上。我在該碩士論文研究中取得的主要結果如下：白化突變體的T-DNA被確定插入於擬南芥的第二染色體上，而該插入點與白化的表現形相連鎖。本人曾嘗試用多種方法克隆有關基因，並已發現若干候選基因。生理學研究顯示該白化突變體的葉綠體無法正常發育，而其光合作用也無法正常進行。該些實驗數據顯示BT20極有可能在葉綠體的生物发生及發育當中扮演重要角色。綜上所述，本人在該碩士論文的研究中發現一全新的、可能受BR調節的葉綠體發育調控因子，對於將來研究BR與葉綠體發育的關係具重要科學價值。 / Chloroplasts are a type of organelles in plants that not only capture light for photosynthesis but are also involved in other important biological processes such as the synthesis of lipids, amino acids and phytohormones. The development and functioning of a chloroplast are coordinated by multiple regulators. Loss of function of any of the regulators may result in abnormal development of chloroplasts and even the whole plant. In my thesis project, I characterized a recessive seedling-lethal albino mutant, named bt20, which was isolated from a genetic mutant screen for bzr1-1D suppressor mutants screen using a T-DNA activation tagging approach in Arabidopsis thaliana. bzr1-1D is a brassinosteroid (BR) hypersensitive mutant that was caused by a dominant mutation in the transcription factor BZR1 in the BR signaling pathway. I confirmed that the T-DNA insertion in the bt20 mutant is located in the chromosome 2 of Arabidopsis and is linked with the albino phenotype. Attempts for the mutant gene cloning have identified several candidate genes but the exact responsible gene(s) remain to be determined. Physiological studies indicated impairment of chloroplasts and photosynthesis within the mutant, suggesting that BT20 gene plays an important role in regulating chloroplast development. We anticipate that our study may lead to the identification of a novel regulator involved in the biogenesis and development of chloroplasts and may establish a molecular link between BR and chloroplast development. / Detailed summary in vernacular field only. / Wong, King Shing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 87-95). / Abstracts also in Chinese. / Thesis/Assessment Committee --- p.i / Statement --- p.ii / Abstract --- p.iii / 摘要 --- p.v / Acknowledgements --- p.vi / Table of Contents --- p.viii / List of Figures --- p.xi / List of Tables --- p.xiii / Chapter Part 1 --- Introduction --- p.1 / Chapter 1.1 --- Brassinosteroids --- p.1 / Chapter 1.1.1 --- Brassinosteroids and its discovery --- p.1 / Chapter 1.1.2 --- Brassinosteroid signal transduction --- p.2 / Chapter 1.1.3 --- The transcription factor BZR1 --- p.5 / Chapter 1.1.4 --- bzr1-1D suppressor mutant screen and the identification of an albino mutant --- p.6 / Chapter 1.2 --- Chloroplast --- p.7 / Chapter 1.2.1 --- Chloroplasts in plants and their origin --- p.7 / Chapter 1.2.2 --- Structure of chloroplasts in plants --- p.8 / Chapter 1.2.3 --- Chloroplast development in cotyledons --- p.11 / Chapter 1.2.4 --- Nuclear gene transcription in chloroplast development --- p.14 / Chapter 1.2.5 --- Protein import for chloroplast development --- p.15 / Chapter 1.2.6 --- Chloroplast gene transcription --- p.16 / Chapter 1.2.7 --- Chloroplast RNA processing and protein translation --- p.18 / Chapter 1.2.8 --- Chloroplast protein processing --- p.19 / Chapter 1.2.9 --- Defects in regulators involving chloroplast development and biogenesis --- p.20 / Chapter 1.2.10 --- Potential relationship between BR and chloroplast development --- p.21 / Chapter Part 2 --- Materials and Methods --- p.22 / Chapter 2.1 --- Plant materials and growing conditions --- p.22 / Chapter 2.2 --- bzr1-1D suppressor mutant screen --- p.23 / Chapter 2.3 --- Genotypic PCR for bzr1-1D background and T-DNA insertion --- p.24 / Chapter 2.4 --- Fresh weight measurement of Arabidopsis plants --- p.26 / Chapter 2.5 --- Genetic crossing of Arabidopsis plants --- p.27 / Chapter 2.6 --- T-DNA insertion site identification --- p.28 / Chapter 2.7 --- Gene Cloning --- p.33 / Chapter 2.8 --- Reverse Transcription PCR (RT-PCR) --- p.36 / Chapter 2.9 --- Gene Cloning and vector constructions --- p.39 / Chapter 2.10 --- Gene transformation into Agrobacteria and Arabidopsis --- p.42 / Chapter 2.11 --- Transgenic plant screen --- p.44 / Chapter 2.12 --- Chlorophyll extraction and measurement --- p.45 / Chapter 2.13 --- Chlorophyll fluorescence assay --- p.46 / Chapter 2.14 --- Electronic microscopy (EM) --- p.48 / Chapter 2.15 --- Brassinolide (BL) and brassinozole (brz) treatments --- p.49 / Chapter Part 3 --- Results --- p.50 / Chapter 3.1 --- Summary of the bzr1-1D suppressor mutant screen --- p.50 / Chapter 3.2 --- Isolation of the albino bt20 mutant from the suppressor mutant screen --- p.52 / Chapter 3.3 --- T-DNA insertion site of bt20 is located in the chromosome 2 of Arabidopsis and is linked to the phenotype --- p.57 / Chapter 3.4 --- Genes flanking the insertion site are over-expressed --- p.59 / Chapter 3.5 --- T-DNA insertion does not cause base pair change in genes flanking the insertion site --- p.60 / Chapter 3.6 --- Over-expression of the selected candidate genes do not reproduce the albino phenotype --- p.62 / Chapter 3.7 --- bt20 seedlings have very low chlorophyll content --- p.64 / Chapter 3.8 --- bt20 has defect in chloroplast development --- p.65 / Chapter 3.9 --- bt20 has very low photosynthesis efficiency --- p.67 / Chapter 3.10 --- The expression of genes encoding plastid proteins in the bt20 mutant --- p.71 / Chapter 3.11 --- bt20 can respond to BL and brz treatment --- p.73 / Chapter 3.12 --- bt20 mutation does not affect the expression of BR biosynthetic gene --- p.75 / Chapter Part 4 --- Discussion --- p.77 / Chapter 4.1 --- bzr1-1D suppressor mutant screen and identification of the bt20 albino mutant --- p.77 / Chapter 4.2 --- Characterization of the bt20 albino mutant and cloning of the BT20 gene --- p.79 / Chapter 4.3 --- The possible role of BT20 role in plant growth and chloroplast development --- p.82 / Chapter 4.4 --- BR can improve the growth of the albino bt20 mutant --- p.85 / Chapter Part 5 --- Conclusion --- p.86 / Chapter Part 6 --- Reference --- p.87
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_328597 |
| Date | January 2013 |
| Contributors | Wong, King Shing., Chinese University of Hong Kong Graduate School. Division of Life Sciences. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | electronic resource, electronic resource, remote, 1 online resource (xiii, 95 leaves) : ill. (some col.) |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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