Maize defective kernel (dek) mutants identify genes necessary for the successful passage of embryos through the embryogenic and maturation phases of embryo development. The goal of this thesis was to characterize the developmental potential of three dek mutants that appeared to be morphologically blocked prior to the maturation phase of embryogenesis. Descriptive and experimental studies of the mutants and their wild-type counterparts were used to compare their morphological and physiological progression through seed development. Parameters of growth, morphogenesis, maturation and germination were measured throughout their ontogeny. Two mutants, cp*-1311C and cp*-1399A, slowly progress to morphological stages 2 and 3, respectively. Growth and maturation processes remain in synchrony with morphology, as indicated by their size, germination behavior and level of storage reserve accumulation. Not every facet of development is retarded. Both dehydration of the seed and the accumulation of desiccation proteins, maize Lea group 3 (MLG3) and Lea group 2 dehydrin (DHN), are more globally regulated, since their accumulation is precocious with respect to embryo morphology. This suggests that some aspects of the embryonic program are mediated by maternal factors. Genetic and developmental characterization of a third mutant, dks8, indicates that it defines a pattern gene that functions to specify the initiation or maintenance of the embryonic shoot. The dks8 mutant is variable in phenotype; mutants with partial and abnormal shoot development are sometimes found on ears segregating for shootless dks8 embryos. dks8 is not allelic to other shootless dek mutants. The dks8 mutation was isolated from an active Mutator transposon stock. RFLP analyses for cosegregation of various Robertson's Mutator transposable elements with the dks8 allele demonstrate that a Mu8 element and dks8 are closely linked. The dks8 allele is under-represented on segregating ears, which may reflect either epigenetic suppression of the mutant phenotype or a bias against the mutant allele in the formation of the female gametophyte. / Graduation date: 1995

No description available.

No description available.

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.

Photocopy of typescript. / Digitized by Kansas Correctional Industries

Typescript (photocopy). / Digitized by Kansas Correctional Industries

Call number: LD2668 .T4 1953 A7 / Master of Science

Thesis (PhD)--Stellenbosch University, 2001. / ENGLISH ABSTRACT: Given the organisation of the maize genome as well as demands placed on the saturation of molecular linkage maps it would be desirable to identify informative molecular markers that is located or linked to genic rich areas. Sequences of gene products from different gene classes were investigated. Proteins containing a nucleotide binding site (NBS) and leucine-rich repeat (LRR) region comprise the largest class of disease resistance proteins. Resistant gene analogue (RGA) primers belonging to this specific class were derived from previous published literature studies. By means of similarity studies of short stretches of conserved amino acid and DNA sequences, primers were developed that belonged to the peroxidase and reductase gene classes. A novel class of transposable element was identified, that occurred in the gene rich areas of a diverse range of grass genomes. Of all the MITE families described so far, the Heartbreaker (Hbr) and Hb2 family elements were of particular interest. The unique properties of MITEs, especially their high copy number, polymorphism, stability and preference for genic areas together with the RGA primers, were exploited to develop a new marker technique for the isolation of a class of molecular marker with a strong preference for genic areas. Using the publicly available recombinant inbred population, Tx303 x C0159, 196 MITE/RGA markers were added to the existing recombinant inbred linkage map consisting of ±1033 already established markers. It became apparent that just like loci for disease resistance, the 196 MITE/RGA fragments were not randomly distributed across the maize genome but occurred in clusters spread across the ten maize chromosomes. Ninety-two (92) of the MITE/RGA fragments showed significant correlation to previously mapped maize resistance genes. To establish the conservation and specificity of both the Hbr and Hb2 elements, sequences of 19 MITE/RGA fragments were ascertained. When comparing the partial MITE element sequences from these fragments, a high degree of element conservation was observed. One fragment showed good sequence correlation to a NADPH He Toxin reductase protein product and mapped to the same chromosomal location as the hm1 gene locus in maize. This fragment can be considered a candidate gene for resistance against the pathogen, Helminthosporium carbonum. The Hbr primer used proved to be very specific for the Heartbreaker MITE element, this was in contrast to the non-specificity of the Hb2 primer. The applicability of this technique was tested on two maize diseases that cause immense damage in the maize production industries in South Africa. Fourteen MITE/RGA markers were used to fine map the putative chromosomal locations for the HtN1, Ht1, Ht2 and Ht3 genes that confer resistance. against Setosphaeria turcica, the northern corn leaf blight (NelS) pathogen in maize. Three MITE/RGA fragments were identified that aided in the saturation of the linkage map for quantitative trait resistance (QTl) against gray leaf spot (GlS) in maize. This novel MITE/RGA technique presented a unique opportunity to search for additional candidate genes by using polymerase chain reaction (peR) analysis. When compared to the conventional amplified fragment length polymorphism (AFLP) technique, the MITE/RGA technique proved to be just as efficient but was more cost effective and less time consuming. / AFRIKAANSE OPSOMMING: Die organisasie van die mielie genoom as ook die vereistes wat daar geplaas word op die versadiging van koppelingskaarte, vereis dat daar meer klem geplaas word op die ontwikkeling van molekulêre tegnieke wat merkers in geenryke areas identifiseer. Die volgordes van geenprodukte, wat behoort tot verskillende geenklasse, is deeglik bestudeer. Proteïenprodukte wat bestaan uit 'n nukleotiedbindingsarea (NBA) en 'n leusienryke herhalende (LRH) area is een van die grootste klasse waaronder siekteweerstandsproteïene sorteer. Polimerase kettingreaksie (PKR) inleiers wat behoort tot hierdie spesifieke klas, is verkry vanuit vorige publikasies. Deur kort gekonserveerde aminosuur en DNS volgordes te vergelyk is inleiers ontwikkel wat behoort tot die peroksidase en reduktase gene klasse. 'n Nuwe klas transponeerbare elemente wat voorkom in die geenryke areas van diverse gras genome, is geïdentifiseer. Van al die miniatuur inversie herhalende transponeerbare elemente (MITE) wat al geïdentifiseer is, is die twee elemente, Heartbreaker (Hbr) en Hb2, van groot belang. Unieke eienskappe van die MITEs, veral hul hoë kopie aantal, polimorfiese-indeks, stabiliteit asook voorkeur vir geenryke areas, tesame met die weerstandsgeen analoë (WGA) inleiers, is gebruik om 'n nuwe merker tegniek te ontwikkel. Hierdie nuwe tegniek identifiseer 'n klas merker wat 'n sterk voorkeur het vir geenryke areas. Deur gebruik te maak van die openbare beskikbare rekombinante ingeteelde (RI) populasie, Tx303 x C0159, is 196 MITE/WGA-merkers gekarteer op die bestaande RIL koppelingskaart, wat alreeds bestaan uit ±1033 gevestigde merkers. Net soos die lokusse vir siekteweerstand het dit geblyk dat hierdie 196 merkers in groepe voorkom wat verspreid is oor die tien mielie chromosome. Twee-en-negentig (92) van die 196 gekarteerde MITE/WGA-merkers het betekenisvolle korrelasie gewys met reeds gekarteerde mielie weerstandsgene. Die volgordes van 19 MITE/WGAfragmente is bepaal om sodoende die spesifisiteit en mate van konservering van die Hbr and Hb2 elemente te bereken. 'n Hoë mate van element konservering is waargeneem. Een fragment het In baie goeie volgorde korrelasie gewys met In NADPH HG toksien reduktase proteïen produk en karteer op dieselfde chromosomale posisie as die hm1 geen lokus. Hierdie fragment kan gesien word as In kandidaatgeen vir weerstand teen die mielie patogeen, Helminthosporium carbonum. Die toepasbaarheid van hierdie tegniek is getoets op twee siekte toestande, wat lei tot groot verliese in die mielie industrie, in Suid-Afrika. Veertien van die MITE/WGAmerkers is gebruik om die waarskynlike chromosomale posisies van die HtN1, Ht1, Ht2 en Ht3 gene, wat weerstand bied teen Setosphaeria turcica, die noordelike mielie blaarvlek (NMBV) patogeen, fyner te karteer. Drie MITE/WGA fragmente is geïdentifiseer wat gehelp het in die versadiging van die koppelingskaart vir die kwantitatiewe kenmerk weerstandbiedenheid (KKW) teen grys blaarvlek (GBV) in mielies. Deur gebruik te maak van polimerase kettingreaksie (PKR) analise, verskaf hierdie tegniek die moontlikheid om te soek vir addisionele kandidaatgene. Hierdie tegniek is ook vergelyk met die konvensionele geamplifiseerde fragment lengte polimorfisme (AFLP) tegniek. Daar is gevind dat die nuwe tegniek net so informatief is, maar wel meer koste effektief en tyd besparend.

This dissertation describes the cloning and characterization of the TOUSLEDlike kinases genes of maize (ZmTLKs). The TOUSLED-like kinases (TLKs) are a conserved family of nuclear Ser/Thr kinases in higher eukaryotes. The maize genome has three TOUSLED-like kinase genes (ZmTLK1, ZmTLK2, and ZmTLK3). Based upon sequence similarity, the ZmTLKs are divided into two classes, the ZmTLK1 and the ZmTLK2/3 class. The origins of these genes can be inferred from their map positions and relationships with TLKs in other Zea species. The ZmTLK1 and ZmTLK2 genes occupy syntenous positions on chromosome arms 1L and 5S in the maize genome. There are two equivalent classes of TLK genes in other Zea species, altogether indicating that the two ZmTLK classes are orthologous genes from the precursor species of maize, an ancient allotetraploid. Gene expression studies of ZmTLKs show that there is a higher level of expression in tissues undergoing DNA synthesis. This is consistent with studies of TLKs in animal systems that show involvement in chromatin assembly/remodeling activities during DNA replication and repair, as well as in transcription. The highest level of gene expression for the ZmTLK2/3 class was observed during development of the endosperm, in a period of massive nuclear endoreduplication. ZmTLK1 is not upregulated in endoreduplicating endosperm, suggesting functional divergence between the two classes of ZmTLK genes. The function of the ZmTLKs was examined by testing whether maize TLK genes could complement the tousled mutant of Arabidopsis. In Arabidopsis thaliana, recessive mutations in the single copy TOUSLED (TSL) gene cause moderate vegetative and severe floral defects, suggesting that TLKs may play a role in gene expression modulation through chromatin remodeling. The ZmTLK proteins are 84% identical to TSL in the catalytic region and 45 - 49% at the N-terminal regulatory domain. However, structural features of the N-terminal region domains of the ZmTLKs are similar to that of TSL. Arabidopsis tsl-1 mutant plants were transformed with ZmTLK2, under the control of the CaMV 35S promoter. These plants showed wild-type Arabidopsis phenotype, indicating that in spite of their sequence differences, ZmTLK2 and TSL interact with the same substrates and regulatory partners and are functionally equivalent. / Graduation date: 2005

D8 originally designated as the dominant stature mutant of maize, was characterized and shown to be incompletely dominant. The study included morphological measurements, cytology, hormone studies, and enzyme and protein analysis. The effect of the D8 mutation can be detected after 40 hours of germination of the coleoptile. Dwarf (D8/d8) seedling length is 1/2 of the normal sib length for coleoptile, first leaf, and mesocotyl. The cell measurements indicate that cell elongation and cell division are involved in the size discrepancy. Mature dwarf plants have shorter internodes and the shorter, wider leaves are a darker green than the normal plant. The homozygous D8/D8 displays normal meiotic division and pollen formation is normal upto the 2 nucleate stage. Pollen viability of the homozygote is low and no seed was obtained in crosses involving this genotype. Several biological stains were used to test pollen viability with the results indicating greater than 85% viability for the heterozygote and less than 15.6% viability for the homozygote. The examination of the pachytene chromosomes of heterozygotes indicates a loop on a large chromosome. This loop is only found in the D8 heterozygote and implies a duplication or deficiency may be involved with the D8 phenotype. Avena straight growth bioassay for auxin displayed no significant difference in auxin production between dwarf and normal coleoptile tips. The D8 dwarf seedlings responded to the exogenous application of auxin, kinetin, and casamino acids in the same pattern as the normal seedlings, but never attained normal stature. Gibberellic acid (GA) and cyclic adenosine monophosphate (cAMP) exogenous applications displayed a difference in dwarf and normal response patterns and implies that the utilization or destruction of these substances may be involved. The investigation of Laemmli gel patterns for the three genotypes failed to show a difference. The soluble proteins formed 27 bands from the coleoptiles of each phenotype. Adh-1 gel patterns and pollen staining was utilized to examine the possibility of a deletion overlapping this locus. The Adh-1 locus has been mapped proximal to the D8 locus. The results indicate the Adh locus is not included in the putative D8 deletion.