三十五肽重複蛋白是線粒體和葉綠體中與RNA轉錄后加工相關的一個家族蛋白。PPR蛋白特異性的和RNA結合，在RNA編輯，剪接，形成成熟的5’端以及蛋白質翻譯等方面起著重要作用。由於PPR蛋白家族很龐大，目前很多PPR蛋白的功能還未知。在這個論文里，我們對兩個三十五肽重複蛋白 PPR1703和 PPR87進行了分子水平上的功能分析。 / PPR1703基因編碼一個含有17個重複結構的P型PPR蛋白。GFP螢光定位的結果顯示，這個蛋白定位於線粒體。爲了研究這個蛋白的功能，我們從UniformMu突變群中分離了ppr1703-1突變體。在ppr1703-1突變體中，PPR1703基因的表達完全喪失。這個基因的突變抑制了胚和胚乳的發育，導致空果皮（emp）表型。這個基因的突變導致部份花粉的發育不良，從而破壞了3:1的分離比。通過比較野生型和突變體線粒體基因的表達發現，nad7第二個內含子的剪接功能在突變體中幾乎喪失。隨後的研究發現，PPR1703基因缺失突變體無法組裝線粒體複合物一，喪失線粒體複合物一活性進而誘導了與交替氧化途徑相關的基因的表達。我們的實驗結果證明，PPR1703基因負責線粒體基因nad7第二個內含子的剪接，並且影響了玉米胚和胚乳的發育。 / PPR87基因編碼一個定位於線粒體的E型PPR蛋白。在PPR87基因缺失型突變體中，有3個線粒體編輯位點的功能消失，他們分別是：NADH脫氫酶1的740編輯位點，NADH脫氫酶7的739編輯位點和膜定位和轉運蛋白的139位點。還有3個編輯位點的功能減低，他們分別是：NADH脫氫酶4L的110位點，膜定位和轉運蛋白的138位點和细胞色素C成熟蛋白亚基的1492位點。在PPR1703突變體中，胚和胚乳的發育受到抑制。我們的工作證明PPR87基因負責多個線粒體RNA位點的編輯，這個基因的突變破壞了線粒體的正常功能，進而影響了種子發育。 / Pentatricopeptide repeat proteins (PPR) are a large family of proteins in land plants with functions implicated in RNA processing in mitochondria and chloroplasts. Each PPR protein is believed to recognize and bind specifically its target sequence in the transcript, performing the function of RNA editing, splicing, 5’ and 3’ end maturation and protein translation regulation. Because of the family size, functions of many PPRs are unknown. In this thesis, we demonstrate the molecular characterization of PPR1703 and PPR87 in maize. / PPR1703 is a P subclass PPR protein, containing 17 PPR repeats. PPR1703-GFP analysis indicated that PPR1703 is targeted to the mitochondrion, which is consistent with bioinformatics prediction. To reveal its function, we isolated a Mutator (Mu) insertional mutant from the UniformMu population in maize, named ppr1703-1. The insertion abolishes the expression of PPR1703, constituting a null allele. The mutant shows severely arrested embryo and endosperm development, causing an empty pericarp phenotype. Its pollen development is partially affected, causing a distortion from 3:1 segregation. Comparative study of the entire mitochondrial transcripts between the WT and the mutant revealed that the nad7 intron 2 splicing is dramatically reduced in the mutant. This deficiency is accompanied by reduced mitochondrial complex I assembly and its activity. These results indicate that PPR1703 is required for mitochondrial nad7 intron 2 splicing and embryogenesis and endosperm development in maize. / PPR87 is an E subclass of the PLS subfamily PPR protein, which was showed to be targeted to mitochondria as well. The Mu-insertional mutant showed embryo and endosperm development arrest at coleoptilar stage. Analysis of the mitochondrial transcripts revealed that loss function of the PPR87 abolishes the C-to-U editing of multiple sites including nad1-740, nad7-739 and mttB(orfX)-139; and also significantly decreases the editing in nad4L-110, mttB(orfX)-138 and ccmFn-1492. These results indicate that PPR87 functions in the C-to-U editing of multiple sites in several transcripts and such editing is essential to the mitochondrial function, thus the embryogenesis and endosperm development in maize. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Yang, Yanzhuo. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 79-98). / Abstracts also in Chinese. / Yang, Yanzhuo.

The Pentatricopeptide Repeat (PPR) gene family produces RNA-binding proteins that target organellar transcripts. The PPR family is expanded in land plants, with nearly 450 genes identified in Arabidopsis thaliana. In plants with a Cytoplasmic Male Sterility (CMS) phenotype, members of the PPR family can act as a RESTORER OF FERTILITY (Rf) and are part of a subset of genes called RESTORER OF FERTILITY-LIKE (RFL). Unlike other PPR transcripts, RFL transcripts are targets of both microRNA (miRNA) and trans-acting siRNA (tasiRNA) and produce secondary siRNA after initial miRNA- or tasiRNA-guided cleavage. We utilized the A. lyrata genome assembly and high-throughput sequencing of small RNA to examine the evolutionary dynamics of the PPR gene family and the pattern of small RNA targeting of RFL transcripts. We found an expanded set of 539 PPR genes in A. lyrata, 51 of which were in the RFL group, often in multiple collinear copies when compared to their A. thaliana orthologs. In-species RFL paralogs appear to be more related to one another than to their collinear orthologs, which is possible evidence of gene conversion or ectopic recombination. miRNA targeting of RFL transcripts is largely conserved with nearly two-thirds of all target sites maintained. TasiRNA targeting was less conserved with roughly one-third of comparable validated tasiRNA targets maintained in both species. However, when clusters of potential tasiRNA targets were considered, roughly two-thirds of target sites are conserved. Production of secondary siRNA from A. lyrata PPR transcripts is less well defined than in A. thaliana, with strong signals coming from phases that are not concordant with the miRNA- or tasiRNA-guided cleavage sites. / Graduation date: 2013