Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: Ribosome inactivating proteins (RIPs) are currently classified as rRNA N-glycosidases, but
also have polynucleotide: adenosine glycosidase activity. RIPs are believed to have anti-viral
and anti-fungal properties, but the exact mechanism of these proteins still need to be
elucidated.The mechanism of resistance however, appears to be independent of the pathogen.
For resistance the RIP terminates virus infected plant cells and stops the reproduction and
spread of the virus. Transgenic plants containing RIPs should thus be resistant to a wide
range of viruses. The ultimate goal of the larger project of which this forms part is the
development of virus resistant plants. To monitor the expression of a RIP in a transgenic
plant a detection method had to be developed. Antibody detection of the RIP was decided
upon as the most cost effective method. The RIP, Dianthin 30 from Dianthus caryophyllus
(carnation), was used and expressed in bacterial and insect expression systems. The bacterial
expression experiments were done using the pET expression system in BL21(DE3)pLysS
cells. The expression in this system yielded recombinant protein at a very low concentration.
Expression experiments were also performed in insect tissue culture with the baculovirus
vector BAC-TO-BAC™.With this system the expression was also too low to be used for the
production of antibodies. A Dianthin 30 specific peptide was then designed and then
produced by Bio-Synthesis. This peptide was then used to raise antibodies to detect Dianthin
30. These antibodies were tested on Dianthus caryophyllus proteins. To establish if this
detection method was effective to monitor the expression in plants, tobacco plants were
transformed with Agrobacterium tumefaciens containing Dianthin 30 in the pART27 plant
expression vector. The putative transformed plants were analysed with peR and Southern
blots. / AFRIKAANSE OPSOMMING: Tans word Ribosomale-inaktiverende proteïene (RIPs) geklassifiseer as rRNA N-glikosidase
wat ook polinukleotied: adenosien glikosidase aktiwiteit bevat. Daar word geglo dat RIPs
anti-virale en anti-fungus eienskappe bevat, maar die meganisme van beskerming word nog
nie ten volle verstaan nie. Dit is wel bewys dat die meganisme van weerstand onafhanklik is
van die patogeen. Virus geinfekteerde plantselle word deur die RIP gedood om die
voortplanting en verspreiding te bekamp en sodoende word weerstand bewerkstellig.
Transgeniese plante wat dan 'n RIP bevat sal dus weerstandbiedend wees teen 'n wye
spektrum virusse. Die hoofdoel van die breër projek, waarvan die projek deel uitmaak: is die
ontwikkeling van virusbestande plante. Om die uitdrukking van die RIP in die transgeniese
plante te kontroleer, moes 'n deteksie metode ontwikkel word. Die mees koste effektiewe
deteksie metode is met teenliggame. Die RIP, Dianthin 30 from Dianthus caryophyllus
(angelier) was gebruik vir uitdrukking in bakteriele- en insekweefselkultuur. Die bakteriele
uitdrukkingseksperimente was gedoen met die pET uitdrukkings sisteem III
BL21(DE3)pLysS selle. Die uitdrukking in die sisteem het slegs rekombinante proteïene
gelewer in uiters lae konsentrasies. Uitdrukkingseksperimente was ook gedoen in
insekweefselkultuur met die baculovirus vektor BAC-To- BACTM. Met die sisteem was die
uitdrukking ook veels te laag om bruikbaar te wees vir die produksie van teenliggame. Daar
is toe 'n peptied ontwerp wat Dianthin 30 kan verteenwoordig vir die produksie van
teenliggame. Die teenliggame is getoets teen Dianthus caryophyllus proteïene. Om vas te stel
of die deteksiemetode wel die uitdrukking van Dianthin 30 sal kan monitor, is tabak ook
getransformeer met Dianthin 30. Die transformasies is gedoen met die hulp van
Agrobacterium tumefaciens en die pART27 plant uitdrukkings vektor. Die plante is getoets
met die polimerase ketting reaksie en Southern klad tegnieke.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/53633 |
| Date | 03 1900 |
| Creators | Maree, H. J. (Hans Jacob) |
| Contributors | Burger, J. T., Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics. Institute for Plant Biotechnology (IPB) |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | 91 pages : illustrations |
| Rights | Stellenbosch University |
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