Abstract
We have constructed a new system for inducible high-level expression of mRNA for foreign genes in transgenic plants by introducing a glucocorticoid-inducible transcription system into the previously developed "mRNA amplification system" where target mRNA can be amplified as a subgenomic RNA by the replicase of a plant tripartite RNA virus, Brome mosaic virus (BMV). In the new amplification system, the amplification of mRNA is tightly regulated by the expression of a subunit of the BMV replicase. Transgenic Nicotiana benthamiana plants (designated GVG1 x 2FR) were produced that contained cDNA of BMV RNA1 coding a subunit of replicase under the control of a tightly regulated, glucocorticoid-inducible promoter. In addition GVG1 x 2FR plants contain cDNAs of BMV RNA2 coding another subunit of the replicase, and a replicable engineered BMV RNA3 derivative (FCP2IFN) carrying the human gamma interferon (IFN) gene under the control of the Cauliflower mosaic virus 35S promoter. When transgenic plants were treated with dexamethasone (DEX), a strong synthetic glucocorticoid, induction of replication and amplification of the 35S-driven FCP2IFN and synthesis of subgenomic mRNA for IFN were observed. Accumulation levels of amplified FCP2IFN were over 300 times higher than those of the 35S-driven FCP2IFN in the GVG1 x 2FR plant without the treatment and those of the mRNA for IFN were 30-230 times higher than in the previous, non-inducible mRNA amplification system. Without DEX treatment, no subgenomic mRNA for IFN was detected in the GVG1 x 2FR plant. The advantages and potential uses of this system are also discussed.
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Claims 5
Claim · model-written summary
The expression system tightly regulated mRNA amplification through replicase-subunit expression.
Not reportedComparatorValue
Verbatim
e0“the amplification of mRNA is tightly regulated by the expression of a subunit of the BMV replicase”
e1“expression of mRNA for foreign genes in transgenic plants”
Claim · model-written summary
Treatment induced transcript amplification and target mRNA synthesis in transgenic plants.
Verbatim
e0“When transgenic plants were treated with dexamethasone (DEX), a strong synthetic glucocorticoid, induction of replication and amplification of the 35S-driven FCP2IFN and synthesis of subgenomic mRNA for IFN were observed”
Claim · model-written summary
The amplified target RNA exceeded untreated levels by over 300-fold.
Verbatim
e0“Accumulation levels of amplified FCP2IFN were over 300 times higher than those of the 35S-driven FCP2IFN in the GVG1 x 2FR plant without the treatment”
Claim · model-written summary
Target mRNA exceeded the earlier system by 30-230-fold.
- Outcome
- mRNA for IFNe0
- Direction
- highere0
- Value
- 30-230 timese0
- Comparator
- previous, non-inducible mRNA amplification systeme0
Not reportedTaxon
Verbatim
e0“mRNA for IFN were 30-230 times higher than in the previous, non-inducible mRNA amplification system”
Claim · model-written summary
Without induction, target mRNA was undetectable.
Not reportedDirection
Verbatim
e0“Without DEX treatment, no subgenomic mRNA for IFN was detected in the GVG1 x 2FR plant”