Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or...
Reexamination Certificate
1999-06-22
2002-05-28
Nelson, Amy J. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
C435S419000, C435S468000, C800S298000, C800S287000, C800S288000
Reexamination Certificate
active
06395962
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to compositions and methods for modulating expression of DNA sequences in plants. Particularly, methods for enhancing gene expression are provided.
BACKGROUND OF THE INVENTION
Plant transformation has produced revolutionary results in plant science. It has in a very short time contributed to the identification and characterization of many new proteins including enzymes and translocators. When investigating the function of a protein in a plant, it is now common to attempt to increase or decrease the expression of the protein by molecular genetic transformation. From the changes effected by transformation, conclusions can be drawn about the role of the corresponding protein in metabolism.
In agriculture, plant genetic engineering has been utilized in many ways to augment protection against pests and to increase the qualitative and quantitative yield of crop plants. New crop species have been developed to increase plant resistance to insects, viruses, and herbicides. For example, transformed tomato plants have been produced to improve the quality in storage properties of tomato fruits, and transformed rapeseed yielding short chain fatty acids have been produced.
Gene transfer is also being utilized to modify the quality of harvested products to maximize their use as food or industrial raw material. In the same manner, attempts to alter the amino acid composition of storage proteins to increase their nutritional value have been reported. These efforts have been met with mixed results.
Transgenic plants are also suitable for producing peptides and proteins used as pharmaceuticals, such as enkephalins, human serum albumins, or interferons. The production in transgenic plants of vaccines for use against various illnesses is being considered to reduce production costs.
Many factors affect gene expression in plants. One mechanism. gene silencing, is an important but little understood regulatory mechanism. The gene silencing phenotype is characterized by reduced levels of the specific mRNA encoded by the suppressed gene. Individual cases of suppression include those in which mRNA level is regulated transcriptionally and those in which it is regulated post-transcriptionally.
Homology-dependent gene silencing is best documented in transgenic plants where it is induced by insertion of multiple copies of homologous transgenes or by insertion of a transgene with homology to an endogenous gene. The phenomenon may also be induced by plant viruses. Recently, it has been reported that gene silencing can be induced by plant virus infections in the absence of any known homology of the viral genome to those genes. It has been proposed that gene silencing may have evolved as a defense mechanism against viral invasion.
Further evidence of a general antiviral defense pathway comes from studies of synergistic viral disease. In plants infected with two viruses, the disease symptoms are more severe than in plants infected with either of the two viruses alone. Many such synergistic diseases involve a member of the potyvirus group of plant viruses. In potyvirus-associated synergisms, the other virus of the pair may be any of a broad range of unrelated viruses. This dramatic increase in host symptoms in doubly infected plants is correlated with an increase in the accumulation of the non-potyvirus. However, there is no corresponding increase or decrease in the level of the potyvirus.
To fully utilize transgenic plants, the mechanisms behind gene expression and suppression need to be controlled. Particularly, methods are needed for modulating gene expression in plants, particularly for enhancing expression. Such methods need to control gene suppression and result in acceptable expression levels of the gene of interest.
SUMMARY OF THE INVENTION
Compositions and methods for modulating gene expression in plants are provided. Compositions comprise the use of a gene silencer (amplicon) in combination with a suppressor sequence (booster or enhancer). Amplicons comprise a targeting sequence corresponding to the gene of interest, the target gene. The amplicon will direct gene silencing of a sequence having homology (the target sequence) to the targeting sequence. The amplicon may optionally comprise a promoter and a sequence that corresponds to at least a part of a viral genome. Enhancers act to suppress post-transcriptional gene silencing, thus boosting or increasing the expression of a target gene. Generally, enhancers comprise proteins encoded by the viral genome that act to suppress gene silencing. In combination, the amplicon and enhancer sequences can be used to boost expression of target sequences in plants. A variety of promoters many be used in the constructs of the invention depending on the desired outcome. Tissue-preferred promoters, inducible promoters. developmental promoters, and the like can be used to direct expression of the target sequence or enhancer sequence in specific tissues and in different developmental stages of the plant.
Transformed plants, plant cells, tissues, and seeds are provided. Such transformed plants, cells, tissues, and seeds exhibit an enhanced expression of target genes or sequences.
DETAILED DESCRIPTION OF THE INVENTION
Compositions and methods for modulating expression of a target sequence in a plant are provided. That is, the expression of the target sequence may be enhanced or decreased. Generally, enhanced expression of the target sequence is effected. By “enhanced expression” is intended that expression of the target sequence is increased over expression observed in conventional transgenic lines for heterologous sequences and over endogenous levels of expression for homologous sequences. Heterologous or exogenous sequences comprise sequences that do not occur in the plant of interest in its native state. Homologous or endogenous sequences are those that are natively present in the plant genome. Generally, expression of the target sequence is increased at least about 25%-50%, preferably about 50%-100%, more preferably about 100%, 200% and greater. The methods of the invention provide for a substantial increase in expression. Yet, this enhanced expression appears to the naked eye to have no observable negative effects on the plant.
The invention relates to gene silencing, specifically the suppression of gene silencing in plants. “Gene silencing” is generally used to refer to suppression of expression of a gene. The degree of reduction may be partial or total reduction in production of the encoded gene product. Therefore, the term should not be taken to require complete “silencing” of expression. Methods for gene silencing are known in the art and include co-suppression and antisense suppression. See, for example, PCT/GB 98/00442 and PCT/GB 98/02862, herein incorporated by reference.
The amplicon comprises a targeting sequence corresponding to the gene of interest, the target gene. The amplicon comprises additional elements including but not limited to a promoter, viral sequences which permit replication, and the like.
PCT/GB98/00442 discloses an amplicon construct to suppress the expression of a target gene. Thus, an amplicon may comprise a transgene DNA construct including a promoter, cDNA of at least part of a viral genome, and a targeting sequence. The targeting sequence is generally foreign to the virus, and acts to specifically target down-regulation of a gene of interest, the target gene. The amplicon comprises a promoter operably linked to a viral replicase, or a promoter sequence operably linked to DNA for transcription in a plant cell of an RNA molecule that includes plant virus sequences that confer on the RNA molecule the ability to replicate in the cytoplasm of a plant cell following transcription. The transcripts replicate as if they are viral RNAs, activating gene silencing.
Thus, the amplicon comprises the targeting sequence (see, for example, PCT/GB 98/02862). In this embodiment, the transient presence of the targeting sequence induces suppression of the target gene in the plant. The silencing is systemic in nat
Alston & Bird LLP
Mehta Ashwin
Nelson Amy J.
University of South Carolina
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