The construction of plant expression vectors for the introduction of leafroll disease resistance in grapevine

[摘要] ENGLISH ABSTRACT:Grapevine leafroll is one of the most damaging viral diseases that affect manyviticultural regions of the world. Numerous reports over the last few yearshave associated closterovirus-like particles with leafroll disease. To date,eight serologically distinct closteroviruses have been isolated from leafrollinfected 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 virusderivedgene is known as pathogen-derived resistance. The viral coat protein(CP) gene, which expresses a structural protein responsible for coating thevirus particles, was used in the first demonstration of virus-derived resistance.Coat protein-mediated resistance is currently the most feasible and mostwidely used method to obtain virus resistance in crop plants.The CP gene of a South African isolate of GLRaV-3 infected grapevine wasisolated, cloned and sequenced. Double stranded RNA (dsRNA) wasextracted from GLRaV-3 infected material and a high molecular weight band,of -18 kb was identified from infected vines. The dsRNA was used as atemplate in a reverse transcription PCR together with GLRaV-3 CP genespecific primers for the amplification of the GLRaV-3 CP gene (975 bp). TheGLRaV-3 CP gene was cloned into the pGem®-T Easy vector. Cloneshosting the CP gene in the sense (pLR3CP+) and antisense (pLR3CP-)orientations respectively were obtained. The sequence obtained from thesetwo clones showed 99.26 % similarity to the only other GLRaV-3 CPnucleotide sequence available. The GLRaV-3 CP gene was excised frompLR3CP+ and pLR3CP- and subcloned into a plant expression vector,pCAMBIA 3301 in the sense (pCamBLR3CP+) and antisense(pCamBLR3CP-) orientations respectively, therefore enabling sense andantisense gene expression in transgenic plants. The GLRaV-3 CP gene wasalso 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 WineBiotechnology, Stellenbosch) for grapevine transformation experiments. Twoof these constructs, pCamBLR3CP+ and pCamBLR3CP- as well as pCAMBIA 3301 were used to transform Nicotiana tabacum by Agrobacteriumtumefaciens-mediated transformation. Plants were selected for their ability towithstand the herbicide, Basta. This resistance is due to the presence of aplant selectable marker gene on each of these constructs, known as the bargene. PCR with GLRaV-3 CP gene specific primers showed no amplificationof the GLRaV-3 CP gene in the plants transformed with pCamBLR3CP+ andpCamBLR3CP-. Southern blot analysis with the GLRaV-3 CP gene ashybridization probe showed no signal for these plants, thus confirming thePCR results. PCR with bar gene specific primers showed no amplification ofthe bar gene in the plants infected with pCAMBIA 3301. The plantstransformed with pCamBLR3CP+ and pCamBLR3CP- were also screened forthe presence of the bar gene. Three of the eight plants tested showedamplification of the -560 bp bar gene. This result suggests that these plantswere transformed with pCAMBIA 3301 (vector without the ligated GLRaV-3CP gene) and not pCamBLR3CP+ or pCamBLR3CP- as had been expected.This project provides preliminary work for the subsequent transformation ofgrapevine with the GLRaV-3 CP gene, in an attempt to impart virusresistance.

[发布机构] Stellenbosch University