The isolation and characterisation of viral promoters for directed expression of transgenes in grapevine
Burger, J T
University of Stellenbosch. Department of Genetics
Waso, H J F
Rybicki, E P
We aim to address the problem of constitutively expressing strangeness in food crops, by switching on a virus resistance gene(s) upon infection by the virus. During the first phase of work, we plan to isolate and characterise some of the promoter elements that drive the subatomic 3-proximal genes of GLRaV-3. We will test the efficacy of this promoter(s) in leafroll infected plant material, using marker genes. Next, to test our hypothesis (if viral RNA polymerises are present, sense transcripts will result, which, in turn, will be translated into functional protein), we plan to express an antisense construct, containing a viral promoter, constitutively. Finally, we plan to replace the marker gene with a resistance gene to GLRaV-3.
Grapevine leafroll is the most destructive disease of grapevines in South Africa. Grapevine leafroll-associated virus 3 (GLRaV-3) is considered the principle pathogen of this disease complex. The introduction of resistance genes by genetic engineering offers the only real long-term solution to the problem. In one of our existing projects, we aim to introduce GLRaV-3 resistance by expressing the viral coat protein (CP) gene in transgenic wine grape cultivars. However, the application of genetic engineering in the agricultural industry, and especially in the food and beverage sectors, has become a very contentious and highly emotional issue. One of the perceived problems is the constitutive expression of foreign proteins in food crops. We propose to circumvent this problem by switching on virus resistance genes only upon infection by the virus, and only in those cells infected by the virus. To this end, we will employ viral promoters to drive transgenes. This project aims to isolate and characterise some of the promoter elements that drive the sub-genomic 3-proximal genes of GLRaV-3. We hope to eventually express an antisense construct comprising a GLRaV-3 promoter/resistance gene, constitutively. This construct will be ‘silent’ under normal circumstances, but should be transcribed into positive sense RNA by the RNA polymerase of an infecting GLRaV-3. These RNA molecules will act as templates for the translation of functional proteins.
Gardner, H and Burger, J T. 2004. Development of GLRaV-3 resistance in grapevine through a viral sub-genomic promoter-driven construct. Paper presented at the 18th Congress of the South African Genetics Society. 4-7 April. Stellenbosch, South Africa.