Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide encodes an inhibitory rna molecule
Reexamination Certificate
1999-11-10
2003-10-21
Mehta, Ashwin (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide encodes an inhibitory rna molecule
C435S320100, C435S419000, C435S468000, C800S280000, C800S288000, C800S298000
Reexamination Certificate
active
06635805
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to “gene silencing” (“gs”) in transgenic plants. It employs “amplicon constructs”, providing in various aspects nucleic acid molecules and vectors, cells and plants containing these, and methods and uses.
BACKGROUND OF THE INVENTION
“Gene silencing” is a term generally used to refer to suppression of expression of a gene. The degree of reduction may be so as to totally abolish production of the encoded gene product, but more usually the abolition of expression is partial, with some degree of expression remaining. The term should not therefore be taken to require complete “silencing” of expression. It is used herein where convenient because those skilled in the art well understand this.
Transgenes may be used to suppress endogenous plant genes. This was discovered originally when chalcone synthase transgenes in petunia caused suppression of the endogenous chalcone synthase genes (29,35). Subsequently it has been described how many, if not all plant genes can be “silenced” by transgenes (11,12,16,17,19,23). Gene silencing requires sequence similarity between the transgene and the gene that becomes silenced (Matzke, M. A. and Matzke, A. J. M. (1995),
Trends in Genetics
, 11: 1-3). This sequence homology may involve promoter regions or coding regions of the silenced gene (Matzke, M. A. and Matzke, A. J. M. (1993)
Annu. Rev. Plant Physiol. Plant Mol. Biol
., 44: 53-76, Vaucheret, H. (1993)
C. R. Acad. Sci. Paris
, 316: 1471-1483, Vaucheret, H. (1994),
C. R. Acad. Sci. Paris
, 317: 310-323, Baulcombe, D. C. and English, J. J. (1996), Current Opinion In Biotechnology, 7: 173-180, Park, Y-D., et al (1996),
Plant J
., 9: 183-194). When coding regions are involved the transgene able to cause gene silencing may have been constructed with a promoter that would transcribe either the sense or the antisense orientation of the coding sequence RNA. In at least one example the coding sequence transgene was constructed without a promoter (Van Blokland, R., et al (1994),
Plant J
., 6: 861-877). It is likely that the various examples of gene silencing involve different mechanisms that are not well understood. In different examples there may be transcriptional or post transcriptional gs (3,4,10,24).
It has also become clear that gene silencing can account for some characteristics of transgenic plants that are not easily reconciled with conventional understanding of genetics. For example the wide variation in transgene expression between sibling lines with a transgene construct is due in part to gene silencing: low expressers are those with a high level of gene silencing whereas high expressers are those in which gene silencing is absent or induced late in plant development (10,13,14). Similarly gene silencing can often explain virus resistance in transgenic lines in which a viral cDNA transgene is expressed at a low level: the gene silencing mechanism acting on RNA inhibits accumulation of both the transgene RNA and the viral RNA (5,10,22).
In principle there is an enormous practical potential of gs for crop improvement. It is possible to silence genes conferring unwanted traits in the plant by transformation with transgene constructs containing elements of these genes. Examples of this type of application include gs of ripening specific genes in tomato to improve processing and handling characteristics of the harvested fruit; gs of genes involved in pollen formation so that breeders can reproducibly generate male sterile plants for the production of F1 hybrids; gs of genes involved in lignin biosynthesis to facilitate paper making from vegetative tissue of the plant; gs of genes involved in flower pigment production to produce novel flower colours; gs of genes involved in regulatory pathways controlling development or environmental responses to produce plants with novel growth habit or (for example) disease resistance; elimination of toxic secondary metabolites by gs of genes required for toxin production. In addition, gs is can be useful as a means of developing virus resistant plants when the transgene is similar to a viral genome.
A major complication in the practical exploitation of this phenomenon to date is the unpredictable and low occurrence of gs. Typically there will be strong gs in as few as 5-20% of lines generated with any one construct (for examples see (28,34)). Therefore, it has not been realistic to attempt gs in plants that are difficult to transform and for which it is difficult to produce many transformants. Similarly, it would be difficult to activate gs against several different traits or against several viruses in the same plant. Even with plants that are easy to transform the need to generate multiple lines limits the ease of exploitation of gs.
The first indication that an inoculated virus could elicit gene silencing was with transgenic plants in which the transgene included cDNA of tobacco etch potyvirus (22). The lines that exhibited this virus-induced gene silencing were initially high level expressers of the transgene. After inoculation with a strain of tobacco etch potyvirus that was identical or highly similar to the transgene there was a reduction in the amount of the transgene RNA and suppression of the originally inoculated virus in the upper leaves of the plant. These upper leaves were described as having recovered because they were virus and symptom free. They were also resistant against a secondary challenge inoculation with virus that was highly similar to the transgene at the nucleotide level. All of these effects are probably due to gs at the post transcriptional level.
However, there is nothing in this report to indicate that virus-induced gene silencing is intrinsically more reproducible than any other type of gene silencing. In fact there were some lines that displayed the virus-induced gene silencing and other lines that did not (22). Furthermore there are many reports that refer to virus inoculation of transgenic plants carrying transgenes that are similar or identical to the inoculated virus. Of these reports only a minority describe virus-induced gene silencing. Thus with viral transgenes the virus induced gene silencing is the exception rather than the rule. That is, as indicated above, there was no indication from this work that gene silencing by inoculated viruses is intrinsically reproducible.
Biosource Technologies, Inc. (20,21) have suggested the use of genetic constructions based on RNA viruses which replicate in the cytoplasm of cells to provide inhibitory RNA, either anti-sense or co-suppressor RNA. Cells are transfected with the cytoplasmically-replicating genetic constructions in which the RNA encoding region is specific for the gene of interest. Experimental evidence illustrating the drawbacks and limitations of the Biosource approach is included below.
SUMMARY OF THE INVENTION
The present invention aims to overcome one or more of the many problems in the art. For instance, experimental evidence included below demonstrates with embodiments of the present invention gs is achievable more reproducibly than with conventional technology.
Briefly, the present invention in various aspects makes use of an amplicon construct which will exhibit gs targeted against sequence with homology to a sequence within the amplicon. An amplicon is a transgene DNA construct including a promoter and cDNA of at least part of a viral genome, and optionally a transcriptional terminator. Preferably, the construct includes a “targeting sequence”, which may be a sequence foreign to the virus, for specifically targeting down-regulation of a gene of interest (“target gene”). Further details are discussed below.
Incorporation of a construct in the genome of transgenic plants in accordance with the present invention may be used to ensure the viral cDNA is transcribed from the promoter in many or most cells of the plant, though use of a tissue- or developmentally-regulated and/or inducible promoter is possible. If the viral cis-acting elements and trans-acting factors necessary for replication are intact there will
Angell Susan Mary
Baulcombe David Charles
Dann Dorfman Herrell and Skillman
Mehta Ashwin
Plant Bioscience Limited
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