Methods for using artifical polynucleotides and compositions thereof to reduce transgene silencing
AssigneeMonsanto Technology LLC
InventorStanislaw FLASINSKI
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Abstract
The materials and methods disclosed provide for polynucleotide molecules sufficiently divergent from polynucleotides naturally contained in plants, or polynucleotides previously introduced into plants as transgenes to permit trait stacking in plant breeding methods or plant transformation methods. The disclosure also provides for methods and compositions to detect the polynucleotides of the invention in plants.
Claims27
A method to reduce transgene silencing in transgenic plants comprising the steps of:
- a) constructing an artificial polynucleotide that is divergent from a known polynucleotide that encodes a substantially identical protein, and
- b) constructing a DNA construct containing said artificial polynucleotide molecule;
- and
- c) transforming said DNA construct into a plant cell;
- and
- d) regenerating said plant cell into a fertile transgenic plant,
- wherein said artificial polynucleotide and said known polynucleotide are divergent if less than 85 percent identical for their entire length and have no polynucleotide sequence lengths more than 23 nucleotides that have 100 percent identity.
An artificial polynucleotide molecule that is divergent from a known polynucleotide that encodes a substantially identical protein, said artificial polynucleotide molecule is selected from the group consisting of:
- SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:35;
- wherein said artificial polynucleotide and said known polynucleotide are divergent if less than 85 percent identical for their entire length and have no polynucleotide sequence lengths more than 23 nucleotides that have 100 percent identity.
A plant cell comprising at least two polynucleotides, wherein said two polynucleotides encode a substantially identical protein and at least one of the polynucleotides is a transgene, and said polynucleotides are less than 85 percent identical in polynucleotide sequence for their entire length and have no polynucleotide sequence lengths more than 23 nucleotides that have 100 percent identity
A DNA molecule selected from the group consisting of:
- SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27.
A plant cell, plant or progeny thereof comprising a DNA molecule selected from the group consisting of:
- SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27.
A pair of DNA molecules comprising a first DNA molecule and a second DNA molecule, wherein the first DNA molecule is SEQ ID NO:24 or its complement and the second DNA molecule is SEQ ID NO:25 or its complement and the pair of DNA molecules when used in a DNA amplification method produces an amplicon diagnostic for an artificial polynucleotide of SEQ ID NO:17.
A pair of DNA molecules comprising a first DNA molecule and a second DNA molecule, wherein the first DNA molecule is SEQ ID NO:26 or its complement and the second DNA molecule is SEQ ID NO:27 or its complement and the pair of DNA molecules when used in a DNA amplification method produces an amplicon diagnostic for an artificial polynucleotide of SEQ ID NO:18.
A DNA detection kit comprising at least one DNA molecule homologous or complementary to a DNA molecule selected from the group consisting of:
- SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27.
A method of detecting the presence of an artificial polynucleotide encoding a glyphosate resistant EPSPS in a DNA sample, the method comprising:
- (a) extracting a DNA sample from a plant;
- and
- (b) contacting the DNA sample with a labeled DNA molecule of sufficient length to be specifically homologous or complementary to an artificial polynucleotide selected from the group consisting of:
- SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:35, wherein said artificial polynucleotide is divergent from a known polynucleotide that encodes a substantially identical glyphosate resistant EPSPS protein, wherein said artificial polynucleotide and said known polynucleotide are divergent if less than 85 percent identical for their entire length and have no polynucleotide sequence lengths more than 23 nucleotides that have 100 percent identity and wherein said labeled DNA molecule is a DNA probe;
- and
- (c) subjecting the sample and DNA probe to stringent hybridization conditions;
- and
- (d) detecting the DNA probe hybridized to the DNA sample.
An isolated polynucleotide that encodes an EPSPS enzyme, wherein said EPSPS enzyme comprises motif SEQ ID NO:34.
A plant cell, plant or progeny thereof tolerant to glyphosate comprising an EPSPS enzyme with motif SEQ ID NO:34
Dependencies are inferred from the claim text.