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.

Zhang, Yafeng. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 53-64). / 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 (M.Tech.: Biotechnology)-Dept. of Biotechnology and Food Technology, Durban University of Technology, 2007 iv, 75 leaves / The development of transgenic plants resulted in the need to utilize the various molecular methods (e.g., ELISA, real - time PCR etc.) for the detection or analysis of the presence or absence of a specific trait in a particular plant (Bt in this study). The overall aim of this study was to optimize a half – seed extraction technique as part of a laboratory protocol for transgenic maize plants and to explore the possibility of using the following molecular techniques: horizontal isoelectric focusing, real - time PCR and ELISA, as methods for detection of the Bt trait for incorporation into the half – seed extraction protocol.

Thesis (M.Tech.: Biotechnology)-Dept. of Biotechnology and Food Technology, Durban University of Technology, 2007 iv, 75 leaves / The development of transgenic plants resulted in the need to utilize the various molecular methods (e.g., ELISA, real - time PCR etc.) for the detection or analysis of the presence or absence of a specific trait in a particular plant (Bt in this study). The overall aim of this study was to optimize a half – seed extraction technique as part of a laboratory protocol for transgenic maize plants and to explore the possibility of using the following molecular techniques: horizontal isoelectric focusing, real - time PCR and ELISA, as methods for detection of the Bt trait for incorporation into the half – seed extraction protocol.

My dissertation research examined the effect of the cultivation of insect-resistant Bacillus thuringiensis (Bt) maize on the soil environment with a goal of understanding how to obtain a balance between technological advancement and maintenance of a healthy soil ecosystem. Although Bt plants may help to reduce pesticide use, conferring benefits to farm workers and the environment, there are still unresolved questions about how the cultivation of Bt plants affects soil organisms. For this dissertation project, I used 14 different genotypes of Bt maize and non-Bt maize (Zea mays) to investigate the effects of transgenic Bt plants on the colonization ability, abundance, and diversity of symbiotic arbuscular mycorrhizal fungi (AMF) in the soil ecosystem over time. My greenhouse studies demonstrated that Bt maize plants exhibited reduced AMF colonization across multiple Bt genotypes and that effects were most pronounced when fertilizer levels were limited and spore density was high. In addition, I found that although differences in AMF colonization between Bt and non-Bt maize were difficult to detect in the field, spore density was reduced in Bt field plots after just one growing season. When I tested the effect of plot history on AMF and plant growth, I found that Bt and non-Bt maize plants had higher leaf chlorophyll content when grown in plots previously cultivated with the same maize line as the previous year, indicative of a positive feedback effect. I also examined potential mechanisms contributing to the reduced AMF colonization observed in Bt maize in greenhouse studies and determined that follow-up experiments should continue to investigate differences in root apoplastic invertase activity and root permeability in Bt and non-Bt maize. Future investigations would also benefit from examining potential differences in root exudate profiles and volatile organic compounds between Bt and non-Bt cultivars. Taken together, my dissertation results suggest that, while difficult to detect in the field, reductions in AMF colonization in Bt maize roots may be ecologically significant as they could lead to a decrease in the abundance of AMF propagules in the soil over time, potentially impacting soil structure and function in areas where Bt crop cultivation is high.

Genetically modified (GM) white and yellow maize, Zea mays, has been commercially released and cultivated in South Africa since 1997/1998. The traits expressed are insect resistance and herbicide tolerance conferred by the bacteria Bacillus thuringiensis (Bt) Cry genes and Agrobacterium 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase gene, respectively. The Cry genes have been used widely to control lepidopteran insect pests but insect resistance to GM Bt crops has been a concern since the introduction of this technology. A management strategy includes refugia planting of 5% non-Bt plants, with no insecticide application, and 20%, where insecticide application is allowed. These refugia are designed to allow the survival of insect pests within restricted planted zones. However, in South Africa there are reports of Bt-resistant stem borer (Busseola fusca) (Fuller) (Lepidoptera: Noctuidae) and non-compliance with refuge planting. The aims of this study were two-fold: 1. To conduct a survey among KwaZulu-Natal (KZN) GM maize growers to ascertain information such as level of compliance with refuge planting and to determine which refugia were predominantly planted and reasons thereof; 2. To conduct a replicated field trial to determine yield, insect borer damage and economic benefit of the 5% unsprayed and 20% sprayed refuge options (including three configurations namely strip, perimeter and block and a 5 and 20% ‘refuge-in-a-bag’ option). The survey indicated that 28 out of 29 (96.6%) KZN Bt maize growers plant the 5% non-sprayed refuge with 27 (96.4%) of those respondents planting the strip configuration for the purpose of insect management (75%) and ease of planting (32.2%). The survey also showed that 7 (seven) i.e. 21.9% of KZN Bt maize growers observed borer damage and although growers are now fully compliant with refugia planting requirements, initially 7 respondents (24.1%) did not comply with or plant refugia correctly. Furthermore, 7 respondents reported insect borer damage in their maize with 4 of the 7 instances (57.1%) likely stemming from incorrectly planted refugia. vii No significant differences in yield or insect damage were observed between the 5 and 20% refugia for any of the planting configurations in the field trial. However due to costs involved with insecticide application and labour required for the operation in the 20% option, these treatments were less economically advantageous than the non-Bt control. The 20% block and strip configurations had a cost benefit ratio of ZAR 7.21 and ZAR 6.67 respectively, earned per R1 spent by the grower compared with ZAR 7.76 in the sprayed control. The cost-benefit comparison for the 5% block and strip configurations was ZAR 8.48 and ZAR 7.71, respectively compared with ZAR 9.44 in the unsprayed control. In addition, the 20% seed mixture limited borer damage to 4.95% when compared with 15.77% damage in the sprayed control (ANOVA, F pr = 0.124). The seed mixtures are not available commercially and the results from the survey indicated that some education and marketing by the seed companies would be advisable prior to their release to the farming community. In order to determine which of the refuge options between 5 and 20% would be more advantageous for growers overall, regardless of the planting configuration; data were grouped and analysed. There were no significant differences in either the yield or insect damage for the 5 and 20% refugia, but the cost-benefit calculations indicated that the 5% option was more cost effective – for the 5 and 20% refugia, ZAR 7.97 and ZAR 7.15 respectively, earned per ZAR 1 spent by the grower (ANOVA, F pr. = 0.03). This is because no insecticide was used in the 5% treatments. Mean ear damage comparisons between the 5 and 20% refugia showed that the 20% refuge in the perimeter configuration incurred the least damage (2.65% ear damage) compared with 5% perimeter (10.86% ear damage), although the reasons for this are not clear. While the results of the field trials showed no significant differences in insect damage and yield with regard to choice of refuge configuration, monitoring insect resistance management remains an integral part of Bt maize crops in South Africa, in order to delay further resistance development and to prolong the viability of Bt technology. / Agriculture and  Animal Health / M. Sc. (Agriculture)

Genetically modified (GM) white and yellow maize, Zea mays, has been commercially released and cultivated in South Africa since 1997/1998. The traits expressed are insect resistance and herbicide tolerance conferred by the bacteria Bacillus thuringiensis (Bt) Cry genes and Agrobacterium 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase gene, respectively. The Cry genes have been used widely to control lepidopteran insect pests but insect resistance to GM Bt crops has been a concern since the introduction of this technology. A management strategy includes refugia planting of 5% non-Bt plants, with no insecticide application, and 20%, where insecticide application is allowed. These refugia are designed to allow the survival of insect pests within restricted planted zones. However, in South Africa there are reports of Bt-resistant stem borer (Busseola fusca) (Fuller) (Lepidoptera: Noctuidae) and non-compliance with refuge planting. The aims of this study were two-fold: 1. To conduct a survey among KwaZulu-Natal (KZN) GM maize growers to ascertain information such as level of compliance with refuge planting and to determine which refugia were predominantly planted and reasons thereof; 2. To conduct a replicated field trial to determine yield, insect borer damage and economic benefit of the 5% unsprayed and 20% sprayed refuge options (including three configurations namely strip, perimeter and block and a 5 and 20% ‘refuge-in-a-bag’ option). The survey indicated that 28 out of 29 (96.6%) KZN Bt maize growers plant the 5% non-sprayed refuge with 27 (96.4%) of those respondents planting the strip configuration for the purpose of insect management (75%) and ease of planting (32.2%). The survey also showed that 7 (seven) i.e. 21.9% of KZN Bt maize growers observed borer damage and although growers are now fully compliant with refugia planting requirements, initially 7 respondents (24.1%) did not comply with or plant refugia correctly. Furthermore, 7 respondents reported insect borer damage in their maize with 4 of the 7 instances (57.1%) likely stemming from incorrectly planted refugia. vii No significant differences in yield or insect damage were observed between the 5 and 20% refugia for any of the planting configurations in the field trial. However due to costs involved with insecticide application and labour required for the operation in the 20% option, these treatments were less economically advantageous than the non-Bt control. The 20% block and strip configurations had a cost benefit ratio of ZAR 7.21 and ZAR 6.67 respectively, earned per R1 spent by the grower compared with ZAR 7.76 in the sprayed control. The cost-benefit comparison for the 5% block and strip configurations was ZAR 8.48 and ZAR 7.71, respectively compared with ZAR 9.44 in the unsprayed control. In addition, the 20% seed mixture limited borer damage to 4.95% when compared with 15.77% damage in the sprayed control (ANOVA, F pr = 0.124). The seed mixtures are not available commercially and the results from the survey indicated that some education and marketing by the seed companies would be advisable prior to their release to the farming community. In order to determine which of the refuge options between 5 and 20% would be more advantageous for growers overall, regardless of the planting configuration; data were grouped and analysed. There were no significant differences in either the yield or insect damage for the 5 and 20% refugia, but the cost-benefit calculations indicated that the 5% option was more cost effective – for the 5 and 20% refugia, ZAR 7.97 and ZAR 7.15 respectively, earned per ZAR 1 spent by the grower (ANOVA, F pr. = 0.03). This is because no insecticide was used in the 5% treatments. Mean ear damage comparisons between the 5 and 20% refugia showed that the 20% refuge in the perimeter configuration incurred the least damage (2.65% ear damage) compared with 5% perimeter (10.86% ear damage), although the reasons for this are not clear. While the results of the field trials showed no significant differences in insect damage and yield with regard to choice of refuge configuration, monitoring insect resistance management remains an integral part of Bt maize crops in South Africa, in order to delay further resistance development and to prolong the viability of Bt technology. / Agriculture and  Animal Health / M. Sc. (Agriculture)