Browsing by Subject "Transgenic plants"
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Item Analysis of transgenic tobacco that express maize catalase3(Texas Tech University, 1999-05) Schake, Sheryl A.Catalases (H2O2: H2O2 oxidoreductase, EC 1.11.1.6; CAT) are hemecontaining tetramers that are important in destroying H2O2 found in different cellular compartments. Maize Cats has been shown to be capable of dismutating H2O2 via either a catalatic or peroxidatic reaction. In addition, increased maize CAT3 transcripts were detected during periods of chilling acclimation. In this study, a maize CatS cDNA was isolated using reverse transcriptase polymerase chain reaction. To better understand the role of maize CAT3 in oxidative stress, we have introduced the transgene that expresses this enzyme into wild type Xanthi NN tobacco (Nicotiana tabacum). Total catalase activities were only slightly higher in transgenic plants as compared to Xanthi NN. While total peroxidatic activity of these transgenic plants was found to be 12-fold higher than in the wild-type tobacco. Thetransgenic Cat3 plants were exposed to various abiotic stresses such as, low temperatures, high temperatures, salinity, chemical treatments, and photooxidation. Increased seedling growth was evident in transgenic seedlings during treatments at low temperatures, high temperatures, and salinity which could implicate increased protection from oxidative damage. No significant protection was evident when transgenic seedlings were treated with methyl viologen or photooxidatlve stress. In addition, lower lipid peroxidation levels in transgenic plants correlated with increased peroxidatic activity in these plants. These data suggests that in wild-type tobacco that express maize CAT3 have increased protection against various forms of oxidative stress.Item Evaluation of Bacillus thuringiensis technology in Texas corn production(Texas Tech University, 2003-12) Youngblood, Jay LeeNot available.Item Evaluation of sucrose phosphate synthase transgenic cotton lines under field conditions in West Texas(Texas Tech University, 2004-05) Hamill, E MargaretNot availableItem Molecular analysis of ascorbate synthesis in plants(Texas Tech University, 2002-05) Hong, KeejongWhile ascorbic acid is a well-known molecule because of its dietary significance, most aspects of its metabolism and some aspects of its function in plants are poorly understood. The ascorbate biosynthetic pathway has not been firmly established even though it reaches millimolecular concentrations in most tissues. Humans and some other animals (including other primates and guinea pigs) depend on ascorbate in their diet because of loss of a functional form of the last enzyme (L-gulono-1,4-lactone dehydrogenase) of the biosynthesis pathway. Thanks to recent advances and new approaches to the investigation of ascorbate biosynthesis and function, we can understand its role in photosynthesis and photoprotection, in defense against ozone and other oxidative stress and possibility about its role in cell division and cell expansion. Studies have shown that ascorbate is critical for scavenging of hydrogen peroxide (H2O2) in and by the finding of ascorbate peroxidase (APX) in plants and Euglena. These results indicate that ascorbate plays a central role in scavenging of H2O2 in plant tissues. On the basis of this research, I attempted to compare the differences in ascorbate synthesis and oxidative stress tolerance in transgenic tobacco plants that express the putative ascorbate biosynthetic enzymes ; L-galactono-1,4- lactone dehydrogenase, L-gulono-1,4-lactone dehydrogenase, L-sorbose dehydrogenase and L-sorbosone dehydrogenase. Tobacco plants that express trans-genes for these enzymes were developed and tested. Plants that express GLDH and GLO showed increased activities of enzymes and increased amount of L-ascorbate. However, analysis of the stress tolerance characteristics of these plants indicates no remarkable increase in protection from oxidative stress. These results, demonstrate that it is possible to manipulate ascorbate biosynthesis in plants, and the role of this increased ascorbate concentration in plants by genetic manipulation against environmental stress. Though the modifications in plants by genetic changes in ascorbate levels are small, it is possible that more optimal approaches could benefit for human nutrition, stress resistance in plants.Item Recovery of Recombinant and Native Proteins from Rice and Corn Seed(2012-02-14) Wilken, Lisa RachellePlants are potential sources of valuable recombinant and native proteins that can be purified for pharmaceutical, nutraceutical, and food applications. Transgenic rice and corn germ were evaluated for the production of novel protein products. This dissertation addresses: 1) the extraction and purification of the recombinant protein, human lysozyme (HuLZ), from transgenic rice and 2) the processing of dry-milled corn germ for the production of high protein germ and corn protein concentrate (CPC). The factors affecting the extraction and purification of HuLZ from rice were evaluated. Ionic strength and pH was used to optimize HuLZ extraction and cation exchange purification. The selected conditions, pH 4.5 with 50 mM NaCl, were a compromise between HuLZ extractability and binding capacity, resulting in 90% purity. Process simulation was used to assess the HuLZ purification efficiency and showed that the processing costs were comparable to native lysozyme purification from egg-white, the current predominant lysozyme source. Higher purity HuLZ (95%) could be achieved using pH 4.5 extraction followed by pH 6 adsorption, but the binding capacity was unexpectedly reduced by 80%. The rice impurity, phytic acid, was identified as the potential cause of the unacceptably low capacity. Enzymatic (phytase) treatment prior to adsorption improved purification, implicating phytic acid as the primary culprit. Two processing methods were proposed to reduce this interference: 1) pH 10 extraction followed by pH 4.5 precipitation and pH 6 adsorption and 2) pH 4.5 extraction and pH 6 adsorption in the presence of TRIS counter-ions. Both methods improved the binding capacity from 8.6 mg/mL to >25 mg/mL and maintained HuLZ purity. Processing of dry-milled corn germ to increase protein and oil content was evaluated using germ wet milling. In this novel method, dry-milled germ is soaked and wet processed to produce higher value protein products. Lab-scale and pilot-scale experiments identified soaking conditions that reduced germ starch content, enhanced protein and oil content, and maintained germ PDI (protein dispersibility index). Soaking at neutral pH and 25 degrees C maintained germ PDI and improved CPC yield from defatted germ flour. CPC with greater than 75% protein purity was produced using protein precipitation or membrane filtration.Item Transformation of cotton plants through an alternate DNA delivery approach(Texas Tech University, 2003-12) Xiang, ZhixinThe objective of this research was to introduce chitinase and â-l,3-glucanase genes into cotton to improve resistance to fungal pathogens through the pollen tube pathway-mediated transformation (PTP). To confirm the success of this method, DNA of a plasmid pRTL2-GUS containing uidA (coding sequence for â-glucuronidase) was introduced into cotton (Gossypium hirsutum L.) 'Paymaster HS26', 'Stovepipe', and 'CA3048'. Seeds formed from treated flowers were allowed to mature on the plants. Seedlings grown from seeds harvested from treated flowers were screened for the presence of uidA by PCR and GUS (â-glucuronidase) expression by histochemical assay. The overall transformation frequency was 11.7 %. Southem hybridizations of T1 progeny confirmed that the uidA transgene was integrated into the cotton genome. GUS expressions were detected in the plants of two generations. In T2 progeny, GUS segregation of uidA followed the expected 3:lMendelian genetic ratio. Our results confirmed transformation of uidA via PTP. Chitinases and â-1,3-glucanases have been used in transgenic plants to improve resistance to pathogens. In this study, fertile, transgenic cotton (Gossypium hirsutum L.) plants expressing a bean chitinase and an Arabidopsis â-l,3-glucanase were generated using pollen tube pathway-mediated transformation system. Ch5b from Phaseolus vulgaris L. and bg2 from Arabidopsis thaliana L. were cloned into plasmid pRTL2 driven by the CaMV 35S promoter. The transgenes ch5b and bg2 were successfully delivered into two breeding lines of cotton. Southern blot and slab blot analyses of DNA isolated from T2 progeny demonstrated that the transgenes were stably integrated into the genome of transgenic cotton plants and inherited by the offspring. Expression of the chitinase and â-1,3-glucanase gene was detected in transgenic T2 cotton plants. Transgenic T2 and T3 plants examined for resistance to the fiingal pathogen Rhizoctonia solani Kuhn in a growth chamber exhibited less root rot and fewer hypocotyl lesions than non-transgenic control plants. Greenhouse screening tests indicated that the ability to resist the fungal pathogen R. solani increased in these plants. Our results suggest that the chitinase and â-l,3-glucanase genes functioned in cotton and were expressed in fertile plants. The pollen tube pathway transformation strategy may be useful for the control of other fungal diseases of plants.