The development of transgenic plants resistant to cucumber mosaic virus and tobacco necrosis virus

 

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dc.contributor.advisor Thomson, Jennifer Ann en_ZA
dc.contributor.advisor Rybicki, Edward P en_ZA
dc.contributor.author Hackland, Andrew F en_ZA
dc.date.accessioned 2016-08-22T12:22:05Z
dc.date.available 2016-08-22T12:22:05Z
dc.date.issued 1994 en_ZA
dc.identifier.citation Hackland, A. 1994. The development of transgenic plants resistant to cucumber mosaic virus and tobacco necrosis virus. University of Cape Town. en_ZA
dc.identifier.uri http://hdl.handle.net/11427/21411
dc.description Bibliography: pages 108-128. en_ZA
dc.description.abstract Cucumber mosaic virus (CMV) and tobacco necrosis virus (TN V) often occur in mixed virus infections in South Africa. Both viruses are of economic importance because of their world-wide distribution, extensive host range and their effects on yields of agriculturally important crop plants. The complete cDNA sequences of CMV-Wemmershoek (CMV-Wem) coat protein (CP) and TNV-F5P CP genes were cloned and subjected to sequence analysis. CMV-Wem is closely related to CMV-WL and CMV-Q, and therefore falls into CMV subgroup II. Similar analysis showed that TNV-F5P is closely related to TNV-A. By characterizing and sequencing these clones the authenticity of the CMV and TNV CP genes was also determined, prior to sub cloning into the appropriate vectors for expression in E. coli and tobacco. Constructs containing both the full-length CP genes of CMV-Wem and TNV-F5P were subcloned in frame with the malE gene, encoding the maltose binding protein (MBP), in the IPTG-inducible pMALTM vector system, and expressed in E. coli. Through immunological detection the authenticity of both CPs was confirmed. The CMV CP translation product expressed in E.coli was used as an antigen to raise antiserum free from contaminating plant host-specific antibodies. The CP genes of both viruses were individually cloned in both orientations (sense and antisense) in Agrobacterium tumefaciens Ti-plasmid-based binary and cointegrate vectors. The study was then extended to include engineering doubly transgenic plants. In order to determine whether the full-length CP is required to mediate virus resistance, a truncated form of the TNV CP was generated by deleting 83 amino acids from the C-terminus. Transgenic Nicotiana tabacum cv Petit Havana SRl plants containing one of a number of different forms of CMV and TNV CP nucleotide sequence were generated. In whole plant studies, mechanical inoculation of Ro lines with CMV-Wem resulted in more than 50% of the CMV CP-sense (CP+) and CP-antisense plants not developing visible systemic disease symptoms. In both the CMV CP+ and doubly transgenic plants CMV-Wem accumulation was delayed, but virus was found to accumulate in the inoculated leaves over time. The CMV CP+ lines showed excellent protection against CMV-Q, but showed only a delay in symptom production when inoculated with CMV -Y, from subgroup I. en_ZA
dc.language.iso eng en_ZA
dc.subject.other Molecular and Cell Biology en_ZA
dc.title The development of transgenic plants resistant to cucumber mosaic virus and tobacco necrosis virus en_ZA
dc.type Doctoral Thesis
uct.type.publication Research en_ZA
uct.type.resource Thesis en_ZA
dc.publisher.institution University of Cape Town
dc.publisher.faculty Faculty of Science en_ZA
dc.publisher.department Department of Molecular and Cell Biology en_ZA
dc.type.qualificationlevel Doctoral
dc.type.qualificationname PhD en_ZA
uct.type.filetype Text
uct.type.filetype Image
dc.identifier.apacitation Hackland, A. F. (1994). <i>The development of transgenic plants resistant to cucumber mosaic virus and tobacco necrosis virus</i>. (Thesis). University of Cape Town ,Faculty of Science ,Department of Molecular and Cell Biology. Retrieved from http://hdl.handle.net/11427/21411 en_ZA
dc.identifier.chicagocitation Hackland, Andrew F. <i>"The development of transgenic plants resistant to cucumber mosaic virus and tobacco necrosis virus."</i> Thesis., University of Cape Town ,Faculty of Science ,Department of Molecular and Cell Biology, 1994. http://hdl.handle.net/11427/21411 en_ZA
dc.identifier.vancouvercitation Hackland AF. The development of transgenic plants resistant to cucumber mosaic virus and tobacco necrosis virus. [Thesis]. University of Cape Town ,Faculty of Science ,Department of Molecular and Cell Biology, 1994 [cited yyyy month dd]. Available from: http://hdl.handle.net/11427/21411 en_ZA
dc.identifier.ris TY - Thesis / Dissertation AU - Hackland, Andrew F AB - Cucumber mosaic virus (CMV) and tobacco necrosis virus (TN V) often occur in mixed virus infections in South Africa. Both viruses are of economic importance because of their world-wide distribution, extensive host range and their effects on yields of agriculturally important crop plants. The complete cDNA sequences of CMV-Wemmershoek (CMV-Wem) coat protein (CP) and TNV-F5P CP genes were cloned and subjected to sequence analysis. CMV-Wem is closely related to CMV-WL and CMV-Q, and therefore falls into CMV subgroup II. Similar analysis showed that TNV-F5P is closely related to TNV-A. By characterizing and sequencing these clones the authenticity of the CMV and TNV CP genes was also determined, prior to sub cloning into the appropriate vectors for expression in E. coli and tobacco. Constructs containing both the full-length CP genes of CMV-Wem and TNV-F5P were subcloned in frame with the malE gene, encoding the maltose binding protein (MBP), in the IPTG-inducible pMALTM vector system, and expressed in E. coli. Through immunological detection the authenticity of both CPs was confirmed. The CMV CP translation product expressed in E.coli was used as an antigen to raise antiserum free from contaminating plant host-specific antibodies. The CP genes of both viruses were individually cloned in both orientations (sense and antisense) in Agrobacterium tumefaciens Ti-plasmid-based binary and cointegrate vectors. The study was then extended to include engineering doubly transgenic plants. In order to determine whether the full-length CP is required to mediate virus resistance, a truncated form of the TNV CP was generated by deleting 83 amino acids from the C-terminus. Transgenic Nicotiana tabacum cv Petit Havana SRl plants containing one of a number of different forms of CMV and TNV CP nucleotide sequence were generated. In whole plant studies, mechanical inoculation of Ro lines with CMV-Wem resulted in more than 50% of the CMV CP-sense (CP+) and CP-antisense plants not developing visible systemic disease symptoms. In both the CMV CP+ and doubly transgenic plants CMV-Wem accumulation was delayed, but virus was found to accumulate in the inoculated leaves over time. The CMV CP+ lines showed excellent protection against CMV-Q, but showed only a delay in symptom production when inoculated with CMV -Y, from subgroup I. DA - 1994 DB - OpenUCT DP - University of Cape Town LK - https://open.uct.ac.za PB - University of Cape Town PY - 1994 T1 - The development of transgenic plants resistant to cucumber mosaic virus and tobacco necrosis virus TI - The development of transgenic plants resistant to cucumber mosaic virus and tobacco necrosis virus UR - http://hdl.handle.net/11427/21411 ER - en_ZA


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