Isolation and characterization of 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) and putrescine N-methyl transferase (PMT) complementary deoxyribonucleic acid (cDNA) in Nicotiana benthamiana using cytoplasmic inhibition of gene expression (CIGE) technology

Date
2006
Authors
Singh, J. Malkeet
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Abstract
The main goal of this thesis is to show that functional genomics studies can be conducted through gene silencing and overexpressing genes via vira1 vectors. This project thus involves the cloning and subcloning of cDNA fragments in the sense and antisense direction for the study of phenotypic changes due to the overexpression and silencing effects of these inserts in N. benthamiana. The cDNAs that are used in this study are DXR (sense), DXR- (antisense), EPSP+ (sense), EPSP- (antisense) and PMT- (antisense). This project will thus seek to confirm the presence of the inserted cDNA fragments in the viral vector at 15 dpi through reverse transcription polymerase chain reaction (RT PCR). This will show the presence of intact recombinant viral vectors containing the cDNA inserts at 15 dpi At some point the cDNA inserts will be deleted causing the recombinant viral vectors to revert back to the wild type. RNA silencing methods can also be used to mimic known herbicides such as norflurozon and a glyphosate compound (RoundupTM) (Monsanto, Minnesota) that inhibits the enzyme EPSP synthase in the shikimate pathway. N. benthamiana DXR was shown to be a new herbicide target to a known antibiotic fosmidomycin which is being represented as an herbicide in this project. Recombinant viral vector containing antisense pmt was made to decrease the levels of nicotine in the N. benthamiana plants. A recombinant viral vector containing the sense dxr insert was also made to overexpress DXR in N. benthamiana to observe its effects on the growth and development of the plant.
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Thesis (M.S.)--University of Hawaii at Manoa, 2006.
Includes bibliographical references (leaves 50-56).
viii, 56 leaves, bound ill. (some col.) 29 cm
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Nicotiana benthamiana -- Genetic engineering
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Theses for the degree of Master of Science (University of Hawaii at Manoa). Molecular Biosciences and Bioengineering; no. 4103
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