Authors:	Naumer, Christian
Title:	Evaluation of a viral vector system, based on a defective interfering RNA of tomato bushy stunt virus, for protein expression and the induction of gene silencing
Online publication date:	23-Jan-2006
Year of first publication:	2006
Language:	english
Abstract:	A viral vector system was developed based on a DI-RNA, a sub-viral particle derived from TBSV-BS3-statice. This newly designed vector system was tested for its applicability in protein expression and induction of gene silencing. Two strategies were pursued. The first strategy was replication of the DI-RNA by a transgenically expressed TBSV replicase and the second was the replication by a so called helper virus. It could be demonstrated by northern blot analysis that the replicase, expressed by the transgenic N. benthamiana plant line TR4 or supplied by the helper virus, is able to replicate DI-RNA introduced into the plant cells. Various genes were inserted into different DI constructs in order to study the vector system with regard to protein expression. However, independent of how the replicase was provided no detectable amounts of protein were produced in the plants. Possible reasons for this failure are identified: the lack of systemic movement of the DI-RNA in the transgenic TR4 plants and the occurrence of deletions in the inserted genes in both systems. As a consequence the two strategies were considered unsuitable for protein expression. The DI-RNA vector system was able to induce silencing of transgenes as well as endogenous genes. Several different p19 deficient helper virus constructs were made to evaluate their silencing efficiency in combination with our DI-RNA constructs. However, it was found that our vector system can not compete with other existing VIGS (virus induced gene silencing) systems in this field. Finally, the influence of DI sequences on mRNA stability on transient GUS expression experiments in GUS silenced plants was evaluated. The GUS reporter gene system was found to be unsuitable for distinguishing between expression levels of wild type plants and GUS silenced transgenic plants. The results indicate a positive effect of the DI sequences on the level of protein expression and therefore further research into this area is recommended. Ein virales Vektorsystem wurde entwickelt basierend auf einer "Defective interfering"-RNS, ein sub-virales Molekül das bei Infektionen von TBSV-BS3-statice vorkommt. Dieses Vektorsystem wurde auf seine Anwendbarkeit in der Proteinexpression und in der Induktion von "Gensilencing" geprüft. Zwei Strategien wurden verfolgt. Die erste Strategie war Replikation der DI-RNS durch eine transgene exprimierte TBSV-Replikase und die zweite war die Replikation durch ein sogenanntes Helfervirus. Es konnte durch Nothernblot Analyse gezeigt werden, daß beide Replikasen in der Lage sind die DI-RNS zu replizieren. Verschiedene Gene wurden in unterschiedliche DI-Konstrukte eingesetzt, um das Vektorsystem hinsichtlich des Proteinexpression zu evaluieren. Jedoch konnte bei beiden Ansätzen kein Protein produziert werden. Als Folge wurden beide Strategien als unpassend für Proteinexpression erachtet. Das DI-RNS Vektorsystem war in der Lage, "silencing" von transgenen sowie endogenen Genen zu verursachen. Jedoch zeigte sich, daß unser Vektorsystem nicht mit anderen vorhandenen Systemen für VIGS (virus induced gene silencing) auf diesem Gebiet konkurrieren kann. Schließlich wurde der Einfluß von DI Sequenzen auf die mRNS Stabilität bei transienten GUS-Expressionen in GUS "gesilenceten" Pflanzen untersucht.
DDC:	570 Biowissenschaften 570 Life sciences
Institution:	Johannes Gutenberg-Universität Mainz
Department:	FB 10 Biologie
Place:	Mainz
ROR:	https://ror.org/023b0x485
DOI:	http://doi.org/10.25358/openscience-3463
URN:	urn:nbn:de:hebis:77-9114
Version:	Original work
Publication type:	Dissertation
License:	In Copyright
Information on rights of use:	https://rightsstatements.org/vocab/InC/1.0/
Appears in collections:	JGU-Publikationen