Methods for using artificial 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.
Claims23
A method to avoid silencing of a transgene comprising SEQ ID NO.16 operably linked to a polynucleotide encoding a chloroplast transit peptide in transgenic plants comprising the steps of:
- a) obtaining a DNA construct comprising an artificial polynucleotide encoding a chloroplast transit peptide operably linked to SEQ ID NO.17
- b) transforming said DNA construct into a plant cell;
- and
- c) regenerating said plant cell into a fertile transgenic plant, wherein said fertile transgenic plant comprises both said artificial polynucleotide and said transgene, and wherein silencing of said artificial polynucleotide and said transgene is avoided.
3-5. (canceled)
An artificial polynucleotide molecule comprising SEQ ID NO:17.
A plant cell comprising at least two polynucleotides, wherein said two polynucleotides encode the same polypeptide and at least one of the polynucleotides is SEQ ID NO:17 operably linked to a polynucleotide encoding a chloroplast transit peptide.
12-13. (canceled)
A plant, seed, cell, plant, or progeny thereof comprising SEQ ID NO:17 operably linked to SEQ ID NO:13.
A method of detecting an artificial polynucleotide comprising SEQ ID NO:17 in a plant cell, plant, or progeny thereof comprising the steps:
- (a) contacting a DNA sample isolated from said plant cell with a pair of DNA molecules that when used in a nucleic acid amplification reaction produces an amplicon that is diagnostic for SEQ ID NO:17;
- (b) performing a nucleic acid amplification reaction, thereby producing the amplicon;
- and
- (c) detecting the amplicon.
A DNA molecule comprising:
- a polynucleotide molecule that specifically hybridizes to SEQ ID NO:17
17-18. (canceled)
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.
21-22. (canceled)
A DNA detection kit comprising at least one DNA molecule of sufficient length to be specifically homologous or complementary to SEQ ID NO:17, wherein said DNA molecule is useful as a DNA probe or DNA primer.
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 and SEQ ID NO:25.
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:17;
- and
- (c) subjecting the sample and DNA probe to stringent hybridization conditions;
- and
- (d) detecting the DNA probe hybridized to the DNA sample.
26-27. (canceled)
A method to avoid transgene silencing in transgenic plants comprising the steps of:
- (a) obtaining said plant cell comprising SEQ ID NO:17;
- and
- (b) regenerating said plant cell into a fertile transgenic plant, wherein said fertile transgenic plant comprises both said polynucleotides, and wherein silencing of said polynucleotides is avoided.
A DNA molecule comprising SEQ ID NO:13.
Dependencies are inferred from the claim text.