Functional analysis of the cellular RNA-directed RNA polymerase (RdRP) in higher plants

The cellular RNA-directed RNA polymerase 2 (RdRP^2) full-length cDNA was successfully cloned and its authentic sequence has been deduced from the Lycopersicon esculentum cultivar Rentita (tomato). Insights into the possible biological function of the RdRP^2 were achieved by down-regulating expression of the endogenous tobacco RdRP^2 using RNA-mediated gene silencing technologies. Eight different RdRP sequence-containing constructs were produced and introduced into the Nicotiana tabacum cv. Petita Havana SR1. Results of the transgenic plants carrying RdRP^2-specific IR and DR transgene constructs indicated that the RdRP^2 could be involved in normal gene regulation. Appearance of phenotypic alterations in a plant line carrying a RdRP^2 DR transgene was indicative of partial down-regulation of the RdRP^2. Absence of any phenotypic alterations in plants carrying a RdRP^1-specific DR transgene construct supported the observations and conclusions that were drawn from plant lines carrying the RdRP^2 IR and DR transgenes. Expression of identical phenotypic alterations in plant lines containing RdRP^2 gene fragments, in either sense or antisense orientation, further supported the essential function of the RdRP^2. The same applies for the observation, that Nicotiana plants could not be infected with the recombinant PVX/RdRP^2. The presented data provided indirect experimental evidence that the RdRP^2 gene was indeed down-regulated by a gene silencing mechanism. These experiments included the generation of transgenic plants that carried RdRP gene constructs comprising highly conserved RdRP regions. Transgenic plants were genotypically characterised by Southern and PCR analysis and for most of them, T-DNA copy numbers as well as arrangements of the integrated DNA could be determined. These plants are now available for further experiments. In the frame of this work, homozygous T1 plant lines containing non-rearranged RdRP^2 constructs as a single copy insert were established. In addition to genotypical examinations, transgene expression levels were investigated by Northern analysis and by PCR amplification with cDNA.

Grantor dc:publisher

Publikationsserver der RWTH Aachen University

Year dc:date

2003

Author dc:creator

• Razvi, Fareha

Contributors dc:contributor

• Fischer, Rainer

• info:eu-repo/classification/ddc/570
• Tomate
• DNS-abhängige-RNS-Polymerasen
• cDNS
• Genklonierung
• DNS-Sequenz
• Biowissenschaften, Biologie
• RdRPs
• PTGS
• RNAi
• Inverted repeat construct
• antisense construct
• sense construct
• 23nucleotides

Statement dc:rights

• info:eu-repo/semantics/openAccess

Language dc:language

eng

Identifier

https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-120792%22

OAI identifier oai:identifier

oai:publications.rwth-aachen.de:58969