An investigation into the high level production of proteins in tobacco using transgenic plants or viral vectors

Bibliography: pages 80-90. === The aim of this project was to construct a high level plant expression vector from the RNA 3 of cucumber mosaic virus strain Y (CMV Y). The 5'- and 3'-untranslated regions (UTRs) of this genome segment were reverse transcribed, cloned and sequenced. The chlor...

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Main Author: Gehringer, Michelle Martine
Other Authors: Rybicki, Edward P
Format: Dissertation
Language:English
Published: University of Cape Town 2016
Subjects:
Online Access:http://hdl.handle.net/11427/19709
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spelling ndltd-netd.ac.za-oai-union.ndltd.org-uct-oai-localhost-11427-197092020-10-06T05:11:07Z An investigation into the high level production of proteins in tobacco using transgenic plants or viral vectors Gehringer, Michelle Martine Rybicki, Edward P Microbiology Bibliography: pages 80-90. The aim of this project was to construct a high level plant expression vector from the RNA 3 of cucumber mosaic virus strain Y (CMV Y). The 5'- and 3'-untranslated regions (UTRs) of this genome segment were reverse transcribed, cloned and sequenced. The chloramphenicol acetyl transferase gene (CAT) was inserted between the two UTRs. This artificial viral cDNA (5'cat3') was cloned immediately downstream of the cauliflower mosaic virus 35 S promoter at the transcription initiation site to make a DNA vector. An RNA vector construct was made by placing the 5'cat3' segment under the control of a T7 RNA promoter sequence. In vitro transcripts, as well as linearised DNA vector constructs were inoculated onto CMV infected plants. Inoculated plants were monitored for CAT expression. No CAT could be detected in total protein extracts of inoculated plants. No CAT mRNA could be detected in northern blots of total RNA extracted from inoculated plants. The vector constructed from the 5' - and 3' -UTR of the RNA 3 of CMV Y did not appear to contain all the necessary attributes for a viral expression vector. To study the expression of a foreign antigen in tobacco, the L 1 capsid protein of human papillomavirus type 16 was cloned into Agrobacterium tumefaciens and used to make transgenic Nicotiana tabacum. Kanamycin resistant tobacco plants were shown to carry the L 1 capsid gene using PCR screening, but western blots on total protein extracts of the transformed plants were indeterminate. Further studies are needed to determine whether the antigen is produced and if it is correctly processed. 2016-05-18T07:13:16Z 2016-05-18T07:13:16Z 1996 Master Thesis Masters MSc http://hdl.handle.net/11427/19709 eng application/pdf University of Cape Town Faculty of Science Department of Molecular and Cell Biology
collection NDLTD
language English
format Dissertation
sources NDLTD
topic Microbiology
spellingShingle Microbiology
Gehringer, Michelle Martine
An investigation into the high level production of proteins in tobacco using transgenic plants or viral vectors
description Bibliography: pages 80-90. === The aim of this project was to construct a high level plant expression vector from the RNA 3 of cucumber mosaic virus strain Y (CMV Y). The 5'- and 3'-untranslated regions (UTRs) of this genome segment were reverse transcribed, cloned and sequenced. The chloramphenicol acetyl transferase gene (CAT) was inserted between the two UTRs. This artificial viral cDNA (5'cat3') was cloned immediately downstream of the cauliflower mosaic virus 35 S promoter at the transcription initiation site to make a DNA vector. An RNA vector construct was made by placing the 5'cat3' segment under the control of a T7 RNA promoter sequence. In vitro transcripts, as well as linearised DNA vector constructs were inoculated onto CMV infected plants. Inoculated plants were monitored for CAT expression. No CAT could be detected in total protein extracts of inoculated plants. No CAT mRNA could be detected in northern blots of total RNA extracted from inoculated plants. The vector constructed from the 5' - and 3' -UTR of the RNA 3 of CMV Y did not appear to contain all the necessary attributes for a viral expression vector. To study the expression of a foreign antigen in tobacco, the L 1 capsid protein of human papillomavirus type 16 was cloned into Agrobacterium tumefaciens and used to make transgenic Nicotiana tabacum. Kanamycin resistant tobacco plants were shown to carry the L 1 capsid gene using PCR screening, but western blots on total protein extracts of the transformed plants were indeterminate. Further studies are needed to determine whether the antigen is produced and if it is correctly processed.
author2 Rybicki, Edward P
author_facet Rybicki, Edward P
Gehringer, Michelle Martine
author Gehringer, Michelle Martine
author_sort Gehringer, Michelle Martine
title An investigation into the high level production of proteins in tobacco using transgenic plants or viral vectors
title_short An investigation into the high level production of proteins in tobacco using transgenic plants or viral vectors
title_full An investigation into the high level production of proteins in tobacco using transgenic plants or viral vectors
title_fullStr An investigation into the high level production of proteins in tobacco using transgenic plants or viral vectors
title_full_unstemmed An investigation into the high level production of proteins in tobacco using transgenic plants or viral vectors
title_sort investigation into the high level production of proteins in tobacco using transgenic plants or viral vectors
publisher University of Cape Town
publishDate 2016
url http://hdl.handle.net/11427/19709
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