This application claims the benefit of U.S. application 60/537,793 filed Jan. 20, 2004, incorporated herein by reference in its entirety.
INCORPORATION OF SEQUENCE LISTING
Two copies of the sequence listing (Seq. Listing Copy 1 and Seq. Listing Copy 2) and a computer-readable form of the sequence listing, all on CD-ROMs, each containing the file named pa_01117.rpt, which is 61,440 bytes (measured in MS-DOS) and was created on Jan. 18, 2005 all of which are incorporated herein by reference.
FIELD OF THE INVENTION
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The invention relates to the field of plant molecular biology and plant genetic engineering and polynucleotide molecules useful for modulating gene expression in plants.
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One of the goals of plant genetic engineering is to produce plants with agronomically desirable characteristics or traits. The proper expression of a desirable transgene in a transgenic plant is one way to achieve this goal. Promoters are non-coding polynucleotide molecules which play an integral role in the overall expression of genes in living cells. Isolated promoters that function in plants are useful for modifying plant phenotypes through the methods of genetic engineering.
Many constitutive promoters are available and are useful for providing good overall gene expression. For example, constitutive promoters such as P-FMV, the promoter from the 35S transcript of the Figwort mosaic virus, (U.S. Pat. No. 6,051,753); P-CaMV 35S, the promoter from the 35S RNA transcript of the Cauliflower mosaic virus, (U.S. Pat. No. 5,530,196); P-Rice Actin 1, the promoter from the actin 1 gene of Oyza sativa, (U.S. Pat. No. 5,641,876); and P-NOS, the promoter from the nopaline synthase gene of Agrobacterium tumefaciens are known to provide some level of gene expression in most or all of the tissues of a plant during most or all of the plant's lifespan. Alternately, many promoters are available with more specific expression patterns such as tissue specificity, temporal specificity, or developmental specificity. These promoters are useful for the targeted expression of a transgene in plants.
Optimal expression of a transgene is useful for producing plants with agronomically desirable characteristics or traits. Such optimal expression often requires a promoter having a specific expression pattern which may not be readily available in known promoters. One example of such a specific expression pattern is a high level of transgene expression in both vegetative and reproductive tissues. The present invention solves this problem by producing novel chimeric promoters containing elements from known promoters. These novel chimeric promoters can then be tested in plants to determine whether the desired expression pattern is indeed achieved.
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In one embodiment the invention provides novel chimeric promoters provided as SEQ ID NO 9-35 comprising a caulimovirus promoter enhancer fused with a plant actin gene promoter and useful for modulating gene expression in plants. In another embodiment the invention provides constructs comprising the novel chimeric promoter and useful for modulating gene expression in plants. In another embodiment the invention provides a transgenic plant comprising the novel chimeric promoter and the seed of the transgenic plant. In another embodiment the invention provides a method of inhibiting weed growth in a field of transgenic glyphosate tolerant crop plants comprising planting the transgenic plants transformed with an expression cassette comprising the novel chimeric promoter operably linked to a DNA molecule encoding a glyphosate tolerance gene and applying glyphosate to the field at an application rate that inhibits the growth of weeds.
BRIEF DESCRIPTION OF THE DRAWINGS
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FIG. 1 represents a section of the CaMV 35S promoter with enhancer domains marked. Also diagrammatically represented are five enhancer domains constructed for use in creating chimeric actin promoters.
FIG. 2 represents the native rice actin 1 promoter and novel chimeric promoters made by fusing the rice actin 1 promoter and selected CaMV 35S promoter enhancer domains.
FIG. 3 represents the native Arabidopsis actin 1 promoter and novel chimeric promoters made by fusing the Arabidopsis actin 1 promoter and selected CaMV 35S promoter enhancer domains.
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The following definitions and methods are provided to better define the present invention and to guide those of ordinary skill in the art in the practice of the present invention. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.
The invention disclosed herein provides novel combinations of polynucleotide molecules for use in constructing novel chimeric promoters. The design, construction, and use of chimeric or hybrid promoters comprising one or more of the enhancer domains of a caulimovirus 35S promoter and a plant actin gene promoter is one object of this invention. The novel chimeric promoter sequences thereof of SEQ ID NO: 9-35, are capable of transcribing operably linked DNA sequences in multiple tissues and therefore can selectively regulate expression of transgenes in multiple tissues.
As used herein, the term “polynucleotide molecule” refers to the single- or double-stranded DNA or RNA of genomic or synthetic origin, i.e., a polymer of deoxyribonucleotide or ribonucleotide bases, respectively, read from the 5′ (upstream) end to the 3′ (downstream) end.
As used herein, the term “polynucleotide sequence” refers to the sequence of a polynucleotide molecule. The nomenclature for DNA bases as set forth at 37 CFR §1.822 is used.
As used herein, the term “gene regulatory activity” refers to the ability to affect transcription or translation of an operably linked transcribable polynucleotide molecule. An isolated polynucleotide molecule having gene regulatory activity may provide temporal or spatial expression or modulate levels and rates of expression of the operably linked transcribable polynucleotide molecule. An isolated polynucleotide molecule having gene regulatory activity may comprise a promoter, intron, leader, or 3′ transcription termination region.
As used herein, the term “gene expression” refers to the transcription of a DNA molecule into a transcribed RNA molecule. Gene expression may be described as related to temporal, spatial, developmental, or morphological qualities as well as quantitative or qualitative indications.
As used herein, the term “regulatory element” refers to a polynucleotide molecule that may affect the transcription or translation of an operably linked transcribable polynucleotide molecule. Regulatory elements such as promoters, leaders, introns, and transcription termination regions are non-coding polynucleotide molecules having gene regulatory activity which play an integral part in the overall expression of genes in living cells. Isolated regulatory elements that function in plants are therefore useful for modifying plant phenotypes through the methods of genetic engineering.
As used herein, the term “promoter” refers to a polynucleotide molecule that is involved in recognition and binding of RNA polymerase II and other proteins (trans-acting transcription factors) to initiate transcription. A plant promoter is a native or non-native promoter that is functional in plant cells. A promoter can be used as a 5′ regulatory element for modulating expression of an operably linked transcribable polynucleotide molecule. Promoters may be defined by their temporal, spatial, or developmental expression pattern.
As used herein, the term “enhancer domain” refers to a cis-acting transcriptional regulatory element, a.k.a. cis-element, which confers an aspect of the overall control of gene expression. An enhancer domain may function to bind transcription factors, trans-acting protein factors that regulate transcription. Some enhancer domains bind more than one transcription factor, and transcription factors may interact with different affinities with more than one enhancer domain. Enhancer domains can be identified by a number of techniques, including deletion analysis, i.e., deleting one or more nucleotides from the 5′ end or internal to a promoter; DNA binding protein analysis using DNase I footprinting, methylation interference, electrophoresis mobility-shift assays, in vivo genomic footprinting by ligation-mediated PCR, and other conventional assays; or by DNA sequence similarity analysis with known cis-element motifs by conventional DNA sequence comparison methods. The fine structure of an enhancer domain can be further studied by mutagenesis (or substitution) of one or more nucleotides or by other conventional methods. Enhancer domains can be obtained by chemical synthesis or by isolation from promoters that include such elements, and they can be synthesized with additional flanking nucleotides that contain useful restriction enzyme sites to facilitate subsequence manipulation. Thus, the design, construction, and use of enhancer domains according to the methods disclosed herein for modulating the expression of operably linked transcribable polynucleotide molecules are encompassed by the present invention
As used herein, the term “chimeric” refers to the product of the fusion of portions of two or more different polynucleotide molecules. As used herein, the term “chimeric promoter” refers to a promoter produced through the manipulation of known promoters or other polynucleotide molecules. Such chimeric promoters may combine enhancer domains that can confer or modulate gene expression from one or more promoters, for example, by fusing a heterologous enhancer domain from a first promoter to a second promoter with its own partial or complete regulatory elements. The novel chimeric promoters of the present invention desirably contain at least one enhancer domain fused to a plant actin promoter. Thus, the design, construction, and use of chimeric promoters according to the methods disclosed herein for modulating the expression of operably linked transcribable polynucleotide molecules are encompassed by the present invention.
As used herein, the term “percent sequence identity” refers to the percentage of identical nucleotides in a linear polynucleotide sequence of a reference (“query”) polynucleotide molecule (or its complementary strand) as compared to a test (“subject”) polynucleotide molecule (or its complementary strand) when the two sequences are optimally aligned (with appropriate nucleotide insertions, deletions, or gaps totaling less than 20 percent of the reference sequence over the window of comparison). Optimal alignment of sequences for aligning a comparison window are well known to those skilled in the art and may be conducted by tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the search for similarity method of Pearson and Lipman, and preferably by computerized implementations of these algorithms such as GAP, BESTFIT, FASTA, and TFASTA available as part of the GCG® Wisconsin Package® (Accelrys Inc., Burlington, Mass.). An “identity fraction” for aligned segments of a test sequence and a reference sequence is the number of identical components which are shared by the two aligned sequences divided by the total number of components in the reference sequence segment, i.e., the entire reference sequence or a smaller defined part of the reference sequence. Percent sequence identity is represented as the identity fraction multiplied by 100. The comparison of one or more polynucleotide sequences may be to a full-length polynucleotide sequence or a portion thereof, or to a longer polynucleotide sequence.
As used herein, the term “substantial percent sequence identity” refers to a percent sequence identity of at least about 80% sequence identity, at least about 90% sequence identity, or even greater sequence identity, such as about 98% or about 99% sequence identity. Thus, one embodiment of the invention is a polynucleotide molecule that has at least about 80% sequence identity, at least about 90% sequence identity, or even greater sequence identity, such as about 98% or about 99% sequence identity with a polynucleotide sequence described herein. Polynucleotide molecules that are capable of regulating transcription of operably linked transcribable polynucleotide molecules and have a substantial percent sequence identity to the polynucleotide sequences of the promoters provided herein are encompassed within the scope of this invention.
Promoter Isolation and Modification Methods
Any number of methods well known to those skilled in the art can be used to isolate fragments of a promoter disclosed herein. For example, PCR (polymerase chain reaction) technology can be used to amplify flanking regions from a genomic library of a plant using publicly available sequence information. A number of methods are known to those of skill in the art to amplify unknown polynucleotide molecules adjacent to a core region of known polynucleotide sequence. Methods include but are not limited to inverse PCR (IPCR), vectorette PCR, Y-shaped PCR, and genome walking approaches. Polynucleotide fragments can also be obtained by other techniques such as by directly synthesizing the fragment by chemical means, as is commonly practiced by using an automated oligonucleotide synthesizer. For the present invention, the polynucleotide molecules were isolated by designing PCR primers based on available sequence information.