High-efficiency silencing of a β-glucuronidase gene in rice is correlated with repetitive transgene structure but is independent of DNA methylation
Wang, Ming-Bo & Waterhouse, Peter M. (2000) High-efficiency silencing of a β-glucuronidase gene in rice is correlated with repetitive transgene structure but is independent of DNA methylation. Plant Molecular Biology, 43(1), pp. 67-82.
Two transgenic callus lines of rice, stably expressing a β-glucuronidase (GUS) gene, were supertransformed with a set of constructs designed to silence the resident GUS gene. An inverted-repeat (i/r) GUS construct, designed to produce mRNA with self-complementarity, was much more effective than simple sense and antisense constructs at inducing silencing. Supertransforming rice calluses with a direct-repeat (d/r) construct, although not as effective as those with the i/r construct, was also substantially more effective in silencing the resident GUS gene than the simple sense and antisense constructs. DNA hybridisation analyses revealed that every callus line supertransformed with either simple sense or antisense constructs, and subsequently showing GUS silencing, had the silence-inducing transgenes integrated into the plant genome in inverted-repeat configurations. The silenced lines containing i/r and d/r constructs did not necessarily have inverted-repeat T-DNA insertions. There was significant methylation of the GUS sequences in most of the silenced lines but not in the unsilenced lines. However, demethylation treatment of silenced lines with 5-azacytidine did not reverse the post-transcriptional gene silencing (PTGS) of GUS. Whereas the levels of RNA specific to the resident GUS gene were uniformly low in the silenced lines, RNA specific to the inducer transgenes accumulated to a substantial level, and the majority of the i/r RNA was unpolyadenylated. Altogether, these results suggest that both sense- and antisense-mediated gene suppression share a similar molecular basis, that unpolyadenylated RNA plays an important role in PTGS, and that methylation is not essential for PTGS.
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|Item Type:||Journal Article|
|Keywords:||Antisense, Direct repeat, DsRNA, Gene silencing, GUS, Inverted repeat, antisense gene, azacitidine, beta glucuronidase gene, callus culture, DNA methylation, gene insertion, gene repression, genetic line, genetic transformation, induced mutation, messenger RNA, repetitive DNA, reverse transcription, rice, transgene, Culture Techniques, DNA, Antisense, DNA, Bacterial, DNA, Plant, Glucuronidase, Oryza sativa, Plants, Genetically Modified, Recombinant Fusion Proteins, Repetitive Sequences, Nucleic Acid, RNA, Plant, Transformation, Genetic, Transgenes, Embryophyta|
|Subjects:||Australian and New Zealand Standard Research Classification > BIOLOGICAL SCIENCES (060000) > GENETICS (060400)
Australian and New Zealand Standard Research Classification > BIOLOGICAL SCIENCES (060000) > PLANT BIOLOGY (060700)
|Divisions:||Current > Schools > School of Earth, Environmental & Biological Sciences
Current > QUT Faculties and Divisions > Science & Engineering Faculty
|Copyright Owner:||Copyright 2000 Springer Netherlands|
|Deposited On:||09 Jan 2014 07:49|
|Last Modified:||09 Jan 2014 07:49|
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