No description available.

Genetic change in Simmental cattle is being accelerated using multiple ovulation and embryo transfer (MOET) techniques. Success of the project depends largely on the ability to generate grade 1 embryos from juvenile heifers following a performance test. Performance testing imposes specific nutritional, management and environmental conditions on heifers which may influence the onset of puberty and subsequent response to superovulation. The objectives of this study were to determine when puberty occurred in Simmental heifers on performance test and to investigate the effects of pubertal development and performance during test on superovulatory response. Heifers were performance tested between 23 and 49 weeks of age. Performance test measurements included: food intake, energy intake, liveweight, backfat depth, muscle depth and muscling score. The onset of puberty was determined in one generation of heifers (n=30) by detecting elevated plasma progesterone levels indicative of first ovulation. Following performance test heifers were superovulated, using ovine FSH, artificially inseminated and embryo recovery carried out at 52 and 61 weeks of age. Puberty occurred in 87% of heifers before the end of test. There was considerable variation in age, liveweight and withers height at puberty, however, daily liveweights gain appeared to be an important determinant of age at puberty. The yield of grade 1 embryos at the first embryo recovery was highest in prepubertal heifers, however, these heifers experienced a substantial decrease in yield at the second recovery apparently due to reduced recovery rates. Investigation of relationships between performance on test and superovulatory response in three generations of heifers (n=110) revealed no significant effect of performance during test on the yield of grade 1 embryos. It was concluded that several factors contributed to reduced embryo yields including: ineffective control of the corpus luteum in cyclic heifers; the incidence of short luteal phases in pre- and peri-pubertal heifers; luteinization of potential ovulatory follicles; and inappropriate timing of exogenous synchrony and superovulation treatments in relation to waves of follicular development.

赤黴素有多種生物學功能，包括促進莖的伸長、種子萌發以及花的發育。玉米赤黴素缺陷型突變體dwarf1 (d1) 表現出植株矮壯和雌雄兩性花，即原為雌花部位發育出雙性花。但是該突變的分子基礎尚不清楚。通過分析多個d1等位基因突變體的分子組成特征，我們證明d1突變體是由能催化赤黴素中間代謝物轉變為活性赤黴素的赤黴素3-氧化酶（ZmGA3ox2） 突變引起的。重組D1 蛋白能於體外催化至少4個反應，包括GA20 轉變為GA3，GA5轉變為GA3，GA20轉變為GA1 以及GA9轉變為GA4等。煙草細胞中D1-GFP 的瞬時表達和細胞組分蛋白質印染分析等兩個獨立的方法，揭示了D1 蛋白是雙定位於細胞核和細胞質中。此結果暗示活性赤黴素能夠在此兩種細胞器中合成。這個雙定位的結果與赤黴素受體GID1蛋白的定位壹致。在早期的玉米雌花發育的過程中，D1 蛋白特異且大量地表達在雌花中的雄花原基細胞，揭示了赤黴素發揮其抑制雄花原基發育的功能需要在該組織大量合成。 / DELLA 蛋白是赤黴素信號傳導的壹個阻遏物。玉米包含僅壹個DELLA蛋白命為DWARF8（D8）。發生在D8 蛋白N端的突變產生了赤黴素非敏感型突變體dwarf8 （d8）。d8突變體與d1突變體有很多共同特征，包括侏儒植株，深綠色的葉片以及雌雄兩性花。這些特點都表明玉米對赤黴素的響應是被DELLA蛋白所抑制的。DELLA蛋白是通過蛋白互作的方式來限制其互作蛋白功能，以達到抑制赤黴素下遊信號傳導的目的。多樣的赤黴素響應必然需要多樣的DELLA互作蛋白。基於赤黴素在調控玉米性別決定過程當中的獨特功能，我們提出假設：玉米當中存在未知的D8互作蛋白。通過酵母雙雜交方法篩選玉米雌花的cDNA文庫，找到14個在酵母系統與D8互作的蛋白。ZmSPX1是這些蛋白中的壹個但不清楚功能。通過雙分子熒光互補實驗和蛋白質體外結合實驗，D8和ZmSPX1之間的互作被進壹步確定。基于此，我们找到数个候选的D8互作蛋白以及证明了ZmSPX1是D8真正的互作蛋白；这些工作都为进一步研究ZmSPX1蛋白在调控性别分化、细胞分裂和分化等赤霉素响应中的作用提供了基础。 / Gibberellins (GA) have multiple biological functions including promoting stem elongation, seed germination and flower development. The GA deficient dwarf1 (d1) mutant in maize displays plant dwarfism and andromonoecy, i.e. forming anthers in the female flower. However, the molecular basis is not clear. Through molecular characterization of multiple d1 alleles, I prove that the d1 is caused by mutations in the GA 3-oxidase (ZmGA3ox2) that converts the inactive GA intermediates to bioactive GAs. The recombinant D1 protein catalyzes at least four reactions in vitro, converting GA20 to GA3, GA5 to GA3, GA20 to GA1 and GA9 to GA4. Subcellular localization analysis by two independent approaches which are in vivo D1-GFP analysis and western blot analysis of organelle fractions revealed that the D1 protein is dual-localized in the nucleus and the cytosol. ZmGA20ox was also localized in both the cytosol and the nucleus by the in vivo GFP fusion analysis. Interestingly, the dual-localization of D1 and ZmGA20ox coincides with the localization of the GA receptor GID1. In early phase of maize female flower development, the D1 protein was found specifically and highly expressed in the stamen primordia within the female florets. These results indicate that bioactive GAs can be synthesized in both the cytosol and the nucleus, and that the suppression of stamen in female florets is mediated by locally synthesized GAs. This finding provides new insights to the understanding of GA biosynthesis and signal transduction in plants. / DELLA proteins are repressors of GA signal transduction. DWARF8 (D8) is a DELLA protein in maize, Mutations in the N-terminal of D8 resulted in dominant GA insensitive dwarf8 (d8) phenotype. d8 displayed similar phenotypes as the d1mutants; i.e. plant dwarfism, dark green leaves and andromonoecy, indicating that D8 is a master repressor mediating these GA functions. DELLA proteins suppressed the downstream signal transduction of GA by restricting their interacting protein functions through protein-protein interaction. Diverse GA responses require numerous DELLA interacting proteins. Based on the unique function of GA in regulating sex determination in maize, I hypothesize that D8 mediates the GA responses by interacting with yet unknown proteins in maize. Through yeast two hybrid screening of the maize ear cDNA library, I identified 14 proteins that showed genuine interaction in yeast system. Among these, a SPX domain containing protein named as ZmSPX1 was present. SPX domain containing proteins in yeast are implicated in cell cycle regulation; however, their functions in plants are unknown. GFP fusion analysis indicated that ZmSPX1 co-localizes with D8 in the nucleus and their interaction was confirmed by bimolecular fluorescence complementation (BiFC) and in vitro pull-down assay. To this point, I have identified several candidates for D8 interacting proteins and provided strong evidence that ZmSPX1 is a bona fide D8 interacting protein which set a foundation for further analysis of its function in mediating GA responses including sex determination, cell division and elongation. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Chen, Yi. / Thesis (Ph.D.) Chinese University of Hong Kong, 2015. / Includes bibliographical references (leaves 60-67). / Abstracts also in Chinese. / Chen, Yi.

Inteins are autocatalytic protein domains that splice out of the nascent polypeptide shortly after translation, requiring no co-factors to facilitate splicing. There is an intein coding sequence within the Prp8 gene of Cryptococcus neoformans, a human pathogen that causes cryptococcosis in immunocompromised people. The intein, Cne PRP8, is a drug target as Prp8 is a central component of the spliceosome and thus believed to be essential to the fungus. Improved knowledge of the intein and its requirements for splicing can contribute to design of a screening system and the search for an inhibitor of intein splicing. Purification of Cne PRP8 for crystallisation was performed using either an N- or a C-terminal His�Tag�, where the N-terminal His�Tag� was removed by 3C protease prior to crystallisation trials. C-terminally His�Tagged� Cne PRP8 formed the largest crystals. The crystals were triangular plates with stepped faces. A 2.8 Å data set was collected with an R[merge] of 0.151 and a mosaicity of 2.1�. A smaller crystal gave a 3.6 Å data set with an R[merge] of 0.085 and a mosaicity of 1.5�. Molecular replacement was not sufficient to solve the structure, likely because the data were weak and the molecules in the asymmetric unit too numerous. Purified Cne PRP8 was additionally shown by circular dichroism to lack regular secondary structure, suggesting that regions of Cne PRP8 could be natively unstructured. Cne PRP8 was expressed as a fusion protein between Haemophilus influenzae trigger factor (HiTF) and a chitin binding domain (CBD). Antibodies to the different parts of the fusion protein facilitated the observation of splicing ability by western blotting. From this it was determined that Cne PRP8 is capable of splicing in a foreign protein context. Context is important, with maximum splicing occurring when Cne PRP8 has two native N-terminal extein residues and one native C-terminal extein residue. The first residue and the last two residues of Cne PRP8 are essential for splicing; additionally the conserved threonine (T62) and histidine (H65) were shown to be catalytically important. Also required for splicing are arginine 154, tyrosine 162, and aspartate 166. Leucine 161 undergoes ~50% splicing when mutated to alanine, and tryptophan 151 undergoes limited C-terminal cleavage, but no splicing, when mutated to alanine. Tryptophan 151 was identified as a potentially crucial residue, which may function to prevent C-terminal cleavage before the N-terminal rearrangements have taken place. Overall it appears that Cne PRP8 residues that are more diverged from the general intein consensus are less essential for splicing. Wild type Cne PRP8 is insensitive to zinc inhibition in vivo. It is also unresponsive to cadmium, calcium, cobalt, lithium, magnesium, manganese and nickel. However, a partially splicing-deficient mutant exhibited further inhibition in response to zinc and cadmium. This mutant also showed a limited increase in splicing efficiency in response to temperatures lower than 37�C. This study has identified critical residues, in addition to those at the splice junctions necessary for catalysis, which participate in splicing intermediates.

Bibliography: leaves 127-151. / xiii, 151, [61] leaves, [19] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / The successful application of genetic engineering in wheat is dependent on the availability of suitable tissue culture and transformation methods. The primary object of this project was the development of these technologies using elite Australian wheat varieties. / Thesis (Ph.D.)--University of Adelaide, Dept. of Plant Science, Waite Campus, 2001?

Bibliography: leaves 103-124. In this project, using rice as a model plant, methods have been developed to improve regeneration frequency and to enhance the efficiency of Agrobacterium mediated transformation technique.

No description available.

No description available.

Plant productivity in many regions of the world is limited primarily as a result of environmental stresses. High temperature and wounding caused by pest and pathogen infection are among the main factors accounting for unpredictable and often severe yield losses worldwide. These stresses, force the plants to alter then gene expression in order to adapt to the changed environment. Attempts were made in the study to isolate and characterise the differentially expressed heat shocked and wound-induced genes to understand the underlying molecular mechanism of heat shock and wounding response. The isolation of the promoters and their use to derive the tissue-specific and high expression of the linked coding sequences will be proved practically more significant. A cDNA clone designated LP 19 was isolated from a differential screening of a cDNA library prepared from lignifying pods of pea line L59. Sequence homology analysis showed that LP19 belongs to the hsp70 gene family. Northern analysis of RNA from pods from pea lines of different genotypes, showing the presence or absence of pod lignification, showed that LP 19 expression was specifically associated with lignification. Several cDNA species derived from transcripts of the LP 19 gene were subsequently isolated, which showed varying positions of poly (A) addition to the 3' untranslated region. Southern blotting of genomic DNA indicated the presence of single gene corresponding to LP 19.The pea hsp70 gene corresponding to LP 19 was isolated from a pea genomic library using LP 19 as a probe. The pea hsp70(LP19) gene predicts an open reading frame encoding a polypeptide of 648 amino acid residues. This sequence is similar to other plant hsp70 proteins. However, unlike most other plant hsp70 genes, the pea hsp70(LP19) gene lacks an intron. 1.8 kb of 5' flanking sequence of hsp70(LP19) gene was also sequenced. The promoter region contains 6 putative consensus heat shock elements (HSEs) as well as 4 A-T rich sites upstream from TATA box. Induction of gene expression of the pea hsp70(LP19) was observed in all organs of the plant after heat shock; the highest level of expression was observed in root, followed by stem and least in leaves. A similar expression pattern for a corresponding gene was observed in chickpea (Cicer arietinum L.). Other stress conditions such as salt stress and wounding failed to induce the expression of hsp70LP(19) gene both in pea and chickpea. The pea hsp70(LP19) promoter region, including 1.8 kb 5'-flanking sequence, and the first 18 amino acids of the coding region, was fused with coding sequence for P- glucuronidase (GUS). Tobacco plants were transformed with this chimaeric gene in order to study tissue specific and developmental expression of the hsp70(LP19) promoter. Histological staining of GUS activity in transgenic tobacco plants showed that protein was present predominantly in the phloem tissue in stem, root and petioles In addition, developmental expression of the hsp70(LP19) gene promoter, without heat shock, was observed in petals, pollen grains, developing seeds as well as in germinating seeds and seedlings at different stages of growth. Quantitative assay of GUS activity by fluorometric assay was used to follow the time course of protein accumulation. Activity was detected within few minutes of the start of heat shock and increased to a maximum after 6 hrs. A high level of GUS activity was observed only in the heat shocked parts of the plant; no endogenous signal that spread systemically from the heat shocked areas to the rest of the plant was observed.Pea and chickpea plants showed a transient increase of polyphenol oxidase (PPO) with maximum level at 48 hrs after wounding. No systemic induction of PPO was observed in unwounded parts in response to both wounding and MeJA treatment. In order to isolate transcripts expressed differentially in response to wounding, a pea subtractive cDNA library was made. 21 subtracted cDNA clones were partially sequenced. Most of the subtracted cDNA clones showed homology with wound or pathogen induced sequences. Northern analysis of the genes corresponding to the subtracted cDNA clones (SC3, SC7, SC12, SC33, SC57 and SC58), indicated differential expression in response to wounding. Full length or nearly full length cDNAs corresponding to 4 subtracted cDNA clones, designated SC10, SC15, SC57 and SC58, were isolated and sequenced. These cDNA clones will be further studied and efforts will be made to isolate their promoters. The tissue-specific expression will be carried out by using promoter-reporter system. These isolated cDNA clones were partially characterised and will be available for further studies to isolate their respective promoters. The tissue specific expression will be carried out by using promoter-reporter system.

No description available.