Hostname: page-component-cd9895bd7-lnqnp Total loading time: 0 Render date: 2024-12-25T05:36:48.389Z Has data issue: false hasContentIssue false

Detailed characterization of the posttranscriptional gene-silencing-related small RNA in a GUS gene-silenced tobacco

Published online by Cambridge University Press:  31 October 2000

GYÖRGY HUTVÁGNER
Affiliation:
BU Genomics, Plant Research International, Wageningen, The Netherlands Agricultural Biotechnology Center, Gödöllő, Hungary
LUDMILA MLYNÁROVÁ
Affiliation:
BU Genomics, Plant Research International, Wageningen, The Netherlands Institute of Plant Genetics and Biotechnology, Nitra, Slovak Republic
JAN-PETER NAP
Affiliation:
BU Genomics, Plant Research International, Wageningen, The Netherlands
Get access

Abstract

Posttranscriptional gene-silencing phenomena such as cosuppression and RNA interference are associated with the occurrence of small, about 21–23 nt short RNA species homologous to the silenced gene. We here show that the small RNA present in silenced transgenic plants can easily be detected in total RNA isolated according to standard procedures. This will allow for the development of routine and early screenings for the presence of small RNA species and, therefore, gene silencing in transgenic plants. We further demonstrate that the small RNA fraction can be visualized with the SYBR Green II RNA stain, isolated from a gel, labeled and used as a specific probe. Using these approaches, we have fine-mapped the sequences of the GUS gene that are represented in the small RNA fraction of a GUS-silenced tobacco line containing an inverted repeat of the GUS gene. In this tobacco line, the silencing-associated small RNA is a mixture of fragments that cover the 3′ two-thirds of the GUS coding region. The 5′ coding and the 3′ noncoding ends of the GUS mRNA are not represented in the small RNA fraction. The RNA fragments are not likely to be a primary synthesis product of an RNA-dependent RNA polymerase, but rather degradation products from nuclease activity. Surprisingly, RNA isolated from wild-type, untransformed plants showed the presence of a similar-sized small RNA pool. This might indicate the existence of small RNA species from putative endogenous genes that are down regulated by a similar posttranscriptional gene-silencing mechanism. The possibility of isolating and labeling the small RNA pool from wild-type plants will provide a way to identify and study such putative genes.

Type
Research Article
Copyright
2000 RNA Society

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)