Browsing by Subject "Gene silencing"

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Molekulare Analyse des Himbeerringflecken Nepovirus (RpRSV) und Herstellung eines Konstrukts zur Induktion von RpRSV?Resistenz in Reben
(2003) Ebel, Rainer; Reustle, Götz
The Raspberry Ringspot Nepovirus (RpRsV) is one of the viruses responsible for the fanleaf disease of grapevine. Two different serological strains of RpRSV exist: the grapevine strain (RpRSV-g) and the cherry strain (RpRSV-ch), which occurs in Germany and Switzerland. RpRSV has two RNAs (RNA1 and RNA2). Both have a genome-linked protein at the 5' ends and are polyadenylated at the 3' ends. RNA1 and RNA2 have one open reading frame flanked by 5' and 3' non-coding regions. The ORFs encode for one large polyprotein which is proteolytically cleaved in smaller functional proteins. The aims of the work were to produce RpRSV grapevine plants and the closer characterisation of RpRSV. The strategy was to create a gene construct, which should induce a gene silencing against the viruses in the grapevine rootstocks. Nicotiana benthamiana was chosen as a test system for the constructs. Because there was no sequence data available both strains were sequenced. The RpRSV strains were propagated in Chenopodium quinoa. The viral RNAs were purified, cDNA synthesized, cloned and sequenced. The sequences were compared and multiple alignments were performed. A sequence from the RNA2 3' non-coding region of RpRSV-ch was selected for the gene construct. An inverted repeat of this sequence was generateted, separated by a plant intron sequence. This construct under the control of a 35S promotor was cloned in the transformation vectors pPZPnptII (antibiotic resistance) and pPZPbar (herbicide resistance). Agrobacterium mediated transformation of Nicotiana benthamiana has been carried out. The T2 generation of the regenerated transgenic tobacco lines were tested for RpRSV resistance. Furthermore full-length clones of the RNA1 and RNA2 of RpRSV-g were produced. Therefore the full-length ds cDNA of both RNA strains were cloned under the control of a 35S promotor. Infection experiments in Chenopodium quinoa with the full clones have successfully been carried out.