The expression of Dianthin 30, a ribosome inactivating protein

[摘要] ENGLISH ABSTRACT:Ribosome inactivating proteins (RIPs) are currently classified as rRNA N-glycosidases, butalso have polynucleotide: adenosine glycosidase activity. RIPs are believed to have anti-viraland anti-fungal properties, but the exact mechanism of these proteins still need to beelucidated.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 andspread of the virus. Transgenic plants containing RIPs should thus be resistant to a widerange of viruses. The ultimate goal of the larger project of which this forms part is thedevelopment of virus resistant plants. To monitor the expression of a RIP in a transgenicplant a detection method had to be developed. Antibody detection of the RIP was decidedupon 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 bacterialexpression experiments were done using the pET expression system in BL21(DE3)pLysScells. The expression in this system yielded recombinant protein at a very low concentration.Expression experiments were also performed in insect tissue culture with the baculovirusvector BAC-TO-BAC™.With this system the expression was also too low to be used for theproduction of antibodies. A Dianthin 30 specific peptide was then designed and thenproduced by Bio-Synthesis. This peptide was then used to raise antibodies to detect Dianthin30. These antibodies were tested on Dianthus caryophyllus proteins. To establish if thisdetection method was effective to monitor the expression in plants, tobacco plants weretransformed with Agrobacterium tumefaciens containing Dianthin 30 in the pART27 plantexpression vector. The putative transformed plants were analysed with peR and Southernblots.

[发布机构] Stellenbosch University