Thesis (MSc)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: Viruses are some of the most important pathogens of grapevines. There are no effective
chemical treatments, and no grapevine- or other natural resistance genes have been discovered
against grapevine infecting viruses. The primary method of grapevine virus control is
prevention by biological indexing and molecular- and serological screening of rootstocks and
scions before propagation. Due to the spread of grapevine viruses through insect vectors, and
in the case of GRSPaV the absence of serological screening, these methods of virus control
are not always effective. In the past several methods, from cross-protection to pathogen
derived resistance (PDR), have been applied to induce plant virus resistance, but with
inconsistent results. In recent years the application of post-transcriptional gene silencing
(PTGS), a naturally occurring plant defense mechanism, to induce targeted virus resistance
has achieved great success. The Waterhouse research group has designed plant
transformation vectors that facilitate specific virus resistance through PTGS. The primary
focus of this study was the production of virus specific transformation vectors for the
introduction of grapevine virus resistance. The Waterhouse system has been successfully
utilised for the construction of three transformation vectors with the pHannibal vector as
backbone. Each vector contains homologous virus coat protein (CP) gene segments, cloned in
a complementary conformation upstream and downstream of an intron sequence. The
primary vector (pHann-SAScon) contains complementary CP gene segments of both GRSPaV
and GLRaV-3 and was designed for the introduction of multiple-virus resistance. For the
construction of the primary vector the GRSPaV CP gene was isolated from RSP infected
grapevines. A clone of the GLRaV-3 CP gene was acquired. The second vector (pHann-
LR3CPsas) contains complementary CP gene segments of GLRaV-3. The third vector
(pHann-LR2CPsas) contains complementary CP gene segments of GLRaV-2. The cassette
containing the complementary CP gene segments of both GRSPaV and GLRaV-3 was cloned
into pART27 (pART27-HSAScon), and used to transform N tabacum cv. Petit Havana
(SRI), through A. tumefaciens mediated transformation. Unfortunately potential
transformants failed to regenerate on rooting media; hence no molecular tests were performed
to confirm transformation. Once successful transformants are generated, infection with a
recombinant virus vector (consisting of PYX, the GFP gene as screenable marker and the
complementary CP gene segments of both GRSPaV and GLRaV-3) will be used to test for the
efficacy of the vectors to induce resistance. A secondary aim was added to this project when
a need was identified within the South African viticulture industry for GRSPaV specific
antibodies to be used in serological screening. To facilitate future serological detection of
GRSPaV, the CP gene was isolated and expressed with a bacterial expression system (pETI4b)
within the E. coli BL2I(DE3)pLysS cell line. The expressed protein will be used to
generate GRSPaV CP specific antibodies. / AFRIKAANSE OPSOMMING: Virusse is van die belangrikste patogene by wingerd. Daar bestaan geen effektiewe chemiese
beheer nie, en geen wingerd- of ander natuurlike weerstandsgene teen wingerdvirusse is al
ontdek nie. Die primêre metode van beheer t.o.v. wingerdvirusse is voorkoming deur
biologiese indeksering, en molekulêre- en serologiese toetsing van onderstokke en entlote
voor verspreiding. As gevolg van die verspreiding van wingerdvirusse deur insekvektore, en
in die geval van GRSPa V die tekort aan serologiese toetsing, is dié metodes van virusbeheer
nie altyd effektief nie. In die verlede is metodes soos kruis-beskerming en patogeen-afgeleide
weerstand (PDR) gebruik om virusweerstand te induseer, maar met inkonsekwente resultate.
In onlangse jare is post-transkripsionele geenonderdrukking (PTGS), 'n natuurlike plantbeskermingsmeganisme,
met groot sukses toegepas om geteikende virusweerstand te
induseer. Die Waterhouse-navorsingsgroep het planttransformasievektore ontwerp wat
spesifieke virusweerstand induseer d.m.v. PTGS. Die vervaardiging van virus spesifieke
tranformasievektore vir die indusering van wingerdvirusweerstand was die primêre doelwit
van hierdie studie. Die Waterhouse-sisteem was gebruik vir die konstruksie van drie
transformasievektore, met die pHannibal vektor as basis. Elke vektor bevat homoloë virus
kapsiedproteïen (CP) geensegmente, gekloneer in 'n komplementêre vorm stroom-op en
stroom-af van 'n intronvolgorde. Die primêre vektor (pHann-SAScon) bevat komplementêre
CP geensegmente van beide GRSPaV en GLRaV-3, en was ontwerp vir die indusering van
veelvoudige-virusweerstand. Die CP-geen van GRSPa V was vanuit RSP-geïnfekteerde
wingerd geïsoleer, vir die konstruksie van die primêre vektor. 'n Kloon van die GLRa V-3
CP-geen was verkry. Die tweede vektor (pHann-LR3CPsas) bevat komplementêre CP
geensegmente van GLRaV-3. Die derde vektor (pHann-LR2CPsas) bevat komplementêre CP
geensegmente van GLRa V-2. Die kasset bestaande uit die komplementêre CP geensegmente
van beide GRSPaV en GLRaV-3, was gekloneer in pART27 (pART27-HSAScon), en gebruik
om N tabacum cv. Petit Havana (SRI) te transformeer d.m.v. A. tumefaciens bemiddelde
transformasie. Ongelukkig het potensiële transformante nie geregenereer op bewortelingsmedia
nie; gevolglik was geen molekulêre toetse gedoen om transformasie te bevestig nie. Na
suksesvolle transformante gegenereer is, sal infeksie met 'n rekombinante-virusvektor
(bestaande uit PYX, die GFP geen as waarneembare merker en die komplementêre CP
geensegmente van beide GRSPa V en GLRa V-3) gebruik word om die effektiwiteit van die
vektore as weerstandsinduseerders te toets. 'n Sekondêre doelwit is by die projek gevoeg toe
'n behoefte aan GRSPaV spesifieke teenliggame binne die Suid-Afrikaanse wynbedryf
geïdentifiseer is, vir gebruik in serologiese toetsing. Om toekomstige serologiese toetsing van
GRSPa V te bemiddel, was die CP-geen geïsoleer en in 'n bakteriële uitdrukkingsisteem
(PETI4b) uitgedruk, in die E. coli BL21(DE3)pLysS sellyn. Die uitgedrukte proteïne sal
gebruik word vir die vervaardiging van GRSPa V CP spesifieke antiliggame.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/53764 |
| Date | 12 1900 |
| Creators | Van Eeden, C. (Christiaan) |
| Contributors | Burger, J. T., Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | 133 p. : ill. |
| Rights | Stellenbosch University |
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