Thesis (MSc)--University of Stellenbosch, 2000. / ENGLISH ABSTRACT: Grapevine leafroll is one of the most damaging viral diseases that affect many
viticultural regions of the world. Numerous reports over the last few years
have associated closterovirus-like particles with leafroll disease. To date,
eight serologically distinct closteroviruses have been isolated from leafroll
infected vines, of which grapevine leafroll associated closterovirus-3
(GLRaV-3) is the best characterized.
Virus resistance in transgenic plants based on the expression of a virusderived
gene is known as pathogen-derived resistance. The viral coat protein
(CP) gene, which expresses a structural protein responsible for coating the
virus particles, was used in the first demonstration of virus-derived resistance.
Coat protein-mediated resistance is currently the most feasible and most
widely used method to obtain virus resistance in crop plants.
The CP gene of a South African isolate of GLRaV-3 infected grapevine was
isolated, cloned and sequenced. Double stranded RNA (dsRNA) was
extracted from GLRaV-3 infected material and a high molecular weight band,
of -18 kb was identified from infected vines. The dsRNA was used as a
template in a reverse transcription PCR together with GLRaV-3 CP gene
specific primers for the amplification of the GLRaV-3 CP gene (975 bp). The
GLRaV-3 CP gene was cloned into the pGem®-T Easy vector. Clones
hosting the CP gene in the sense (pLR3CP+) and antisense (pLR3CP-)
orientations respectively were obtained. The sequence obtained from these
two clones showed 99.26 % similarity to the only other GLRaV-3 CP
nucleotide sequence available. The GLRaV-3 CP gene was excised from
pLR3CP+ and pLR3CP- and subcloned into a plant expression vector,
pCAMBIA 3301 in the sense (pCamBLR3CP+) and antisense
(pCamBLR3CP-) orientations respectively, therefore enabling sense and
antisense gene expression in transgenic plants. The GLRaV-3 CP gene was
also subcloned from pCamBLR3CP+ into another plant expression vector,
pCAMBIA 2301 in the sense orientation and designated as pCVSLR3CP+.
These three constructs were given to Dr. M. Vivier (Institute for Wine
Biotechnology, Stellenbosch) for grapevine transformation experiments. Two
of these constructs, pCamBLR3CP+ and pCamBLR3CP- as well as pCAMBIA 3301 were used to transform Nicotiana tabacum by Agrobacterium
tumefaciens-mediated transformation. Plants were selected for their ability to
withstand the herbicide, Basta. This resistance is due to the presence of a
plant selectable marker gene on each of these constructs, known as the bar
gene. PCR with GLRaV-3 CP gene specific primers showed no amplification
of the GLRaV-3 CP gene in the plants transformed with pCamBLR3CP+ and
pCamBLR3CP-. Southern blot analysis with the GLRaV-3 CP gene as
hybridization probe showed no signal for these plants, thus confirming the
PCR results. PCR with bar gene specific primers showed no amplification of
the bar gene in the plants infected with pCAMBIA 3301. The plants
transformed with pCamBLR3CP+ and pCamBLR3CP- were also screened for
the presence of the bar gene. Three of the eight plants tested showed
amplification of the -560 bp bar gene. This result suggests that these plants
were transformed with pCAMBIA 3301 (vector without the ligated GLRaV-3
CP gene) and not pCamBLR3CP+ or pCamBLR3CP- as had been expected.
This project provides preliminary work for the subsequent transformation of
grapevine with the GLRaV-3 CP gene, in an attempt to impart virus
resistance. / AFRIKAANSE OPSOMMING: Wingerd rolblaar is een van die mees beskadigende virale siektes wat baie
wingerd areas in die wêreld aantas. In Aantal verslae oor die afgelope jare
het closterovirus partikels met wingerd rolblaar geassosieer. Tot hede, is agt
serologiese onderskeibare closterovirusse geïsoleer vanuit geaffekteerde
wingerde, waarvan wingerd rolblaar geassosieerde closterovirus-3 (GLRaV-3)
die beste gekarakteriseerd is.
Virus bestandheid in transgeniese plante gebaseer op die uitdrukking van
gene afkomstig vanaf virusse, staan bekend as patogeen-afgeleide
weerstand. Die virale kapsule protein (CP) geen vervaardig In strukturele
protein wat verantwoordelik is vir die bedekking van die virus partikel. Dié
geen was gebruik in die eerste demonstrasie van patogeen-afgeleide
weerstand. Kapsuul protein-bemiddelde weerstand is tans die mees praktiese
en algemene gebruikte metode om virus weerstand in plant gewasse te
verkry. Die CP geen van In Suid Afrikaanse isolaat van GLRaV-3
geïnfekteerde wingerde is geïsoleer, gekloneer en die volgorde is bepaal.
Dubbelstring RNA (dsRNA) was uit GLRaV-3 geïnfekteerde materiaal
geëkstraheer en In hoë molekulêre gewig band van -18 kb is geïdentifiseer.
Die dsRNA is gebruik as In templaat vir In omgekeerde transkripsie PKR
saam met GLRaV-3 CP geen spesifieke inleiers vir die amplifikasie van die
GLRaV-3 CP geen (975 bp). Die GLRaV-3 CP geen is gekloneer in die
pGem®-T Easy vektor. Klone met die CP geen in die sin (pLR3CP+) en
teensin (pLR3CP-) oriëntasies respektiewelik is verkry. Die volgorde wat
verkry is vanuit hierdie twee klone dui op In 99.26 % ooreenstemming met die
enigste ander GLRaV-3 CP geen volgorde wat beskikbaar is. Die GLRaV-3
CP geen is uit pLR3CP+ en pLR3CP- gesny en is gesubkloneer in In plant
ekspressie vektor, pCAMBIA 3301 in die sin (pCamBLR3CP+) en teensin
(pCamBLR3CP-) oriëntasies respektiewelik, wat die sin en teensin geen
ekspressie in transgeniese plante in staat stel. Die GLRaV-3 CP geen was
ook gesubkloneer vanaf pCamBLR3CP+ in In ander plant ekspressie vektor,
pCAMBIA 2301 in die sin orientasie en is as pCVSLR3CP+ benoem. Hierdie
drie konstruksies is aan Dr. M. Vivier (Instituut vir Wyn Biotegnologie,
Stellenbosch) gegee vir wingerd transformasie eksperimente. Twee van hierdie konstruksies, pCamBLR3CP+ en pCamBLR3CP- asook pCAMBIA
3301 is gebruik om Nicotiana tabacum deur middel van Agrobacterium
tumefaciens-bemiddelde transformasie te transformeer. Plante is geselekteer
vir hul vermoë om die onkruiddoder, Basta, te weerstaan. Die
teenwoordigheid van die plant selekteerbare merker geen, bar, op elke
konstruksie lui tot dié weerstand. Die plante wat getransformeer is met
pCamBLR3CP+ en pCamBLR3CP- is deur PKR saam met die GLRaV-3 CP
geen spesifieke inleiers getoets, en geen amplifikasie van die GLRaV-3 CP
geen is getoon nie. Southern blot analise met die GLRaV-3 CP geen as
hibridisasie peiler het geen sein gewys vir hierdie plante nie, wat die PKR
resultate bevestig. Die plante wat getransformeer is met pCAMBIA 3301 is
deur PKR saam met die bar geen spesifieke inleiers getoets, en geen
amplifikasie van die bar geen is getoon nie. Die plante wat getransformeer is
met pCamBLR3CP+ en pCamBLR3CP- is ook getoets vir die
teenwoordigheid vir die bar geen. Drie van die agt plante wat getoets is, het
amplifikasie van die -560 bp bar geen getoon. Hierdie onverwagte resultate
stel voor dat dié plante met pCAMBIA 3301 (vektor sonder die geligeerde
GLRaV-3 CP geen) en nie met pCamBLR3CP+ en pCamBLR3CPgetransformeer
is nie. Hierdie projek verskaf voorlopige werk vir die
daaropvolgende transformasie van wingerd met die GLRaV-3 CP geen in 'n
poging om virus bestandheid te verskaf.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/51950 |
| Date | 12 1900 |
| Creators | Van Straten, Celene Debra |
| 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 | Unknown |
| Type | Thesis |
| Format | xviii, 124 p. : ill. |
| Rights | Stellenbosch University |
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