种子发育是开花植物生命周期中的一个关键阶段。理解控制这一过程的机理既是植物生物学的重要基础研究课题，也是农业应用上的重要任务。因为种子是我们的主食，而种子质量和大小正是主要由那些控制种子发育过程的基因控制着。玉米作为一种典型的单子叶植物，是很好的研究单子叶植物种子发育的模式植物。 / 在开花植物中，RNA编辑是发生在线粒体和质体中的一种转录后机制，主要将特定的胞嘧啶转换为尿嘧啶。这一过程很多时候将改变基因组的遗传信息。这是一种非常重要的转录后调节机制，许多案例表明它对编码蛋白的功能十分关键。例如，RNA编辑能修复一个保守的氨基酸密码，创造一个起始或终止密码子，或移除一个终止密码子而编码出一个更大的功能蛋白。因此，RNA编辑不能正常进行可能损害或完全失去编码蛋白的功能，对植物的生长和发育造成严重后果。 / 本研究中，我们报道了玉米种子突变体emp5(empty pericarp 5)的分子特征研究。Emp5基因的无效突变导致玉米种子胚和胚乳发育在早期发育阶段停止。Emp5基因编码一个定位在线粒体的PPR-DYW蛋白。对emp5突变体线粒体转录组的分析表明，EMP5蛋白功能失效阻碍了rpl16-458（野生型中100%被编辑）这一位点的C-to-U RNA编辑，降低了nad9, cox3和rps12这三个转录本中总共9个位点的C-to-U RNA编辑水平。令人意外的是，同时也增加了atp6, nad1, cob 和 rpl16 4个转录本中共5个位点的RNA编辑水平。EMP5蛋白缺失E+和DYW结构域仍然保留了底物的特异性和RNA编辑功能，只是编辑效率有所降低。这表明EMP5蛋白的E+和DYW结构域对其编辑功能不关键，但对编辑效率是必需的。对EMP5在水稻中的同源蛋白的分析表明，OsEMP5在水稻线粒体rpl16-458位点的编辑功能是保守的。OsEMP5的基因沉默表达导致水稻植株生长缓慢及种子缺陷。这些结果表明Emp5基因编码的这一PPR-DYW蛋白对多个线粒体转录本的RNA编辑是必需的。尤其是rpl16-458这一位点的编辑对线粒体的功能十分重要，因而对玉米种子发育非常关键。 / Seed development is a critical stage in the life cycle of flowering plants. Understanding the mechanism governing this process is both a fundamental question in plant biology and also an important task in agriculture application as seeds are staple food and seed quality and size are controlled by the genes governing seed development process. Maize as a typical monocot plant, is also an excellent model system for monocot seed development research. / In flowering plants, RNA editing is a post-transcriptional mechanism that converts specific cytidines to uridines in both mitochondrial and plastidial transcripts, altering the genetic information encoded by these genes. It is important for posttranscriptional regulation and in some cases critical to the functions of the encoded proteins. For example, editing can restore a conserved amino acid codon, create an initiation or stop codon, or remove a stop codon that leads to a functional larger protein. Therefore, deficiency in editing may result in a compromised or complete loss of function for the encoded proteins, leading to a severe consequence in plant growth and development. / In this study, we report the molecular characterization of the empty pericarp 5 (emp5) mutant in maize (Zea mays). Null mutation of Emp5 results in abortion of embryo and endosperm development at early stages. Emp5 encodes a mitochondrion targeted DYW-subgroup PPR protein. Analysis of the mitochondrial transcripts reveals that loss of the EMP5 function abolishes the C-to-U editing of rpl16-458 (100% edited in the wildtype), decreases the editing at nine sites in nad9, cox3 and rps12, and surprisingly increases the editing at five sites of atp6, nad1, cob and rpl16. EMP5 lacking the E+ and DYW domain still retains the substrate specificity and editing function, only at reduced efficiency. This suggests that the E+ and DYW domains of EMP5 are not essential to the EMP5 editing function, but necessary for efficiency. Analysis of the ortholog in rice indicates that OsEMP5 has a conserved function in C-to-U editing of the rice mitochondrial rpl16-458 site. Knock-down expression of OsEmp5 results in slow growth seedlings and defective seeds. These results demonstrate that EMP5 encodes a PPR-DYW protein that is required for the editing of multiple transcripts in mitochondria and the editing events, particularly the C-to-U editing at the rpl16-458 site, are critical to the mitochondrial functions and hence to seed development in maize. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Liu, Yujun. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 78-86). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese. / Thesis/Assessment Committee --- p.i / Statement --- p.ii / Acknowledgements --- p.iii / Abstract --- p.iv / 摘要 --- p.vi / Table of Contents --- p.viii / List of Tables --- p.xii / List of Figures --- p.xiii / List of Abbreviations --- p.xv / Chapter Chapter 1. --- General Introduction --- p.1 / Chapter Chapter 2. --- Literature Review --- p.6 / Chapter 2.1. --- Seed development in flowering plants --- p.7 / Chapter 2.1.1 --- Seed morphogenesis of flowering plants --- p.7 / Chapter 2.1.2 --- Molecular mechanisms of seed development --- p.8 / Chapter 2.2 --- PPR protein family --- p.11 / Chapter 2.2.1 --- Definition of PPR protein --- p.11 / Chapter 2.2.2 --- Subgroups of PPR protein --- p.12 / Chapter 2.2.3 --- PPR protein distribution and evolution --- p.13 / Chapter 2.2.4 --- Functions of PPR proteins in plants --- p.14 / Chapter 2.3 --- PPR proteins involving in seed development --- p.15 / Chapter 2.4 --- PPR proteins studied in maize --- p.17 / Chapter 2.5 --- RNA processing in plant mitochondria --- p.19 / Chapter 2.5.1 --- RNA splicing --- p.20 / Chapter 2.5.2 --- 5’ and 3’ processing of plant mitochondrial transcrips --- p.23 / Chapter 2.5.3 --- RNA stabilization --- p.24 / Chapter 2.6 --- RNA editing --- p.25 / Chapter 2.7 --- Status of C to U RNA editing mechanism --- p.28 / Chapter Chapter 3. --- Materials and methods --- p.31 / Chapter 3.1 --- Plant Materials --- p.32 / Chapter 3.2 --- Light Microscopy of Cytological Sections --- p.32 / Chapter 3.3 --- Immunohistochemistry Analysis Materials --- p.32 / Chapter 3.4 --- Isolation of genomic DNA Materials --- p.33 / Chapter 3.5 --- Southern Analysis Materials --- p.34 / Chapter 3.6 --- Inverse PCR cloning Materials --- p.35 / Chapter 3.7 --- RNA Extraction and RT-PCR Materials --- p.35 / Chapter 3.8 --- Subcellular Localization of EMP5 Protein Materials --- p.36 / Chapter 3.9 --- Analysis of Mitochondrial RNA Editing Materials --- p.37 / Chapter 3.10 --- Rice Transformation Materials --- p.37 / Chapter 3.10.1 --- OsEmp5 RNAi vector construction Materials --- p.37 / Chapter 3.10.2 --- Callus induction from mature rice seeds Materials --- p.38 / Chapter 3.10.3 --- Callus subculture Materials --- p.38 / Chapter 3.10.4 --- Preparation of Agrobacterium tumefaciens Materials --- p.38 / Chapter 3.10.5 --- Co-cultivation Materials --- p.39 / Chapter 3.10.6 --- Callus washing and selection Materials --- p.39 / Chapter 3.10.7 --- Regeneration Materials --- p.40 / Chapter 3.10.8 --- Screening of transgenic plants Materials --- p.40 / Chapter Chapter 4. --- Results --- p.42 / Chapter 4.1 --- Phenotypic and Genetic Characterization of emp5-1 --- p.43 / Chapter 4.2 --- Cloning of Emp5 --- p.45 / Chapter 4.3 --- Emp5 Encodes a Mitochondrion-Targeted PPR-DYW Subclass Protein --- p.48 / Chapter 4.4 --- Expression of Emp5 --- p.51 / Chapter 4.5 --- EMP5 is Required for Mitochondrial RNA Editing --- p.52 / Chapter 4.6 --- Molecular Characterization of emp5-4 Allele --- p.54 / Chapter 4.7 --- Functional Analysis of the Rice OsEmp5 Gene --- p.58 / Chapter Chapter 5. --- Discussion --- p.62 / Chapter 5.1 --- Abortion of emp5-1 Mutant Seed Development is Caused by Defective Mitochondrial RNA Editing --- p.63 / Chapter 5.2 --- Increased Editing in the emp5 Mutant --- p.65 / Chapter 5.3 --- Substrate Specifying Sequence of EMP5 Are Not Conserved --- p.66 / Chapter 5.4 --- The E+ and DYW Motif of EMP5 Is not Essential for This Protein Function --- p.67 / Chapter Chapter 6. --- Conclusion --- p.75 / References --- p.78
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_328215 |
| Date | January 2013 |
| Contributors | Liu, Yujun, 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 (xv, 86 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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