Methods of controlling gene expression and gene silencing
OwnerJoshua Levin, Kenneth Phillips +3inventors
InventorsJoshua Levin, Kenneth Phillips, Gregory Budziszewski, Frederick Meins JR., Evgueni A. Glazov
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Abstract
The present invention relates to methods to regulate gene expression in plants. In particular, manipulation of the expression in a plant cell of a nucleotide sequence encoding a polypeptide comprising a 3′-5′ exonuclease domain is disclosed. More stable and predictable expression is thus obtained. The present invention also relates to method of increasing or decreasing post-transcriptional silencing. The invention further relates to novel nucleic acid molecules comprising nucleotide sequences encoding polypeptides comprising a 3′-5′ exonuclease domain.
Claims59
An isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide comprising a 3′-5′ exonuclease domain, and wherein said polypeptide is identical or substantially similar to an amino acid sequence of SEQ ID NO:
- 2, SEQ ID NO:
- 4, SEQ ID NO:
- 6, SEQ ID NO:
- 22, SEQ ID NO:
- 10, SEQ ID NO:
- 12, or SEQ ID NO:
- 24.
An isolated nucleic acid molecule comprising a nucleotide sequence=identical or substantially similar to SEQ ID NO:
- 1, SEQ ID NO:
- 3, SEQ ID NO:
- 5, SEQ ID NO:
- 21, SEQ ID NO:
- 9, SEQ ID NO:
- 11, or SEQ ID NO:
- 13, or SEQ ID NO:
- 23.
An isolated nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO:
- 23 or complements thereof.
An isolated nucleic acid molecule consisting of the nucleotide sequence of SEQ ID NO:
- 23 or complement thereof.
An expression cassette comprising a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide comprising a 3′-5′ exonuclease domain, and wherein said polypeptide is identical or substantially similar to an amino acid sequence of SEQ ID NO:
- 2, SEQ ID NO:
- 4, SEQ ID NO:
- 6, SEQ ID NO:
- 22, SEQ ID NO:
- 10, SEQ ID NO:
- 12, SEQ ID NO:
- 16, SEQ ID NO:
- 18, or SEQ ID No:
- 24, a promoter, and a terminator.
An isolated and substantially purified polypeptide comprising the amino acid sequence of SEQ ID NO:
- 24.
An isolated and substantially purified polypeptide consisting of the amino acid sequence of SEQ ID NO:
- 24.
A plant cell comprising an endogenous nucleotide sequence identical or substantially similar to SEQ ID NO:
- 1, SEQ ID NO:
- 3, SEQ ID NO:
- 5, SEQ ID NO:
- 21, SEQ ID NO:
- 9, SEQ ID NO:
- 11, SEQ ID NO:
- 13 or SEQ ID NO:
- 23, and wherein said plant cell comprises a mutation in said endogenous nucleotide sequence, or in a regulatory region thereof.
A plant cell capable of expressing a sense RNA molecule of a nucleotide sequence identical or substantially similar to SEQ ID NO:
- 1, SEQ ID NO:
- 3, SEQ ID NO:
- 5, SEQ ID NO:
- 7, SEQ ID NO:
- 21, SEQ ID NO:
- 9, SEQ ID NO:
- 11, SEQ ID NO:
- 13, or SEQ ID NO:
- 23, and an anti-sense RNA molecule of said nucleotide sequence identical or substantially similar to SEQ ID NO:
- 1, SEQ ID NO:
- 3, SEQ ID NO:
- 5, SEQ ID NO:
- 7, SEQ ID NO:
- 21, SEQ ID NO:
- 9, SEQ ID NO:
- 11, SEQ ID NO:
- 13 or SEQ ID NO:
- 23, wherein said sense and said anti-sense RNA molecules are capable of forming a double-stranded RNA molecule.
A method for altering the expression in a plant cell or plant of an endogenous nucleotide sequence encoding a polypeptide comprising a 3′-5′ exonuclease domain, wherein said polypeptide is identical or substantially similar to SEQ ID NO:
- 2, SEQ ID NO:
- 4, SEQ ID NO:
- 6, SEQ ID NO:
- 22, SEQ ID NO:
- 10, SEQ ID NO:
- 12 or SEQ ID NO:
- 24 comprising the step of:
- altering the transcription or translation of said endogenous nucleotide sequence in said plant cell or plant.
A method for altering the expression of an endogenous nucleotide sequence that is identical or substantially similar to SEQ ID NO:
- 1, SEQ ID NO:
- 3, SEQ ID NO:
- 5, SEQ ID NO:
- 21, SEQ ID NO:
- 9, SEQ ID NO:
- 11, SEQ ID NO:
- 13 or SEQ ID NO:
- 23 in a plant cell or plant comprising the step of introducing into said plant cell a means for altering the transcription or translation of said endogenous nucleotide sequence in said plant cell.
A method for altering the expression of a nucleotide sequence of interest in a plant cell or plant comprising the steps of:
- a) altering the expression in said plant cell or plant of an endogenous nucleotide sequence of said plant cell that is identical or substantially similar to SEQ ID NO:
- 1, SEQ ID NO:
- 3, SEQ ID NO:
- 5, SEQ ID NO:
- 21, SEQ ID NO:
- 9, SEQ ID NO:
- 11, SEQ ID NO:
- 13 or SEQ ID NO:
- 23;
- and
- b) introducing into said plant cell or plant a nucleic acid molecule comprising said nucleotide sequence of interest, wherein the expression of said nucleotide sequence of interest in said plant cell or plant is altered.
A method for stabilizing the expression of a nucleotide sequence of interest in a plant cell or plant comprising the steps of:
- a) obtaining a plant cell or plant having altered expression in a plant cell of an endogenous nucleotide sequence of said plant cell or plant that encodes a polypeptide comprising a 3′-5′ exonuclease domain, and wherein said polypeptide is identical or substantially similar to an amino acid sequence of SEQ ID NO:
- 2, SEQ ID NO:
- 4, SEQ ID NO:
- 6, SEQ ID NO:
- 22, SEQ ID NO:
- 10, SEQ ID NO:
- 12, SEQ ID NO:
- 14, or SEQ ID NO:
- 24;
- and
- b) introducing into said plant cell or plant a nucleotide sequence of interest, wherein the expression of said nucleotide sequence of interest in said plant cell is stabilized as compared to the expression of said nucleotide sequence of interest in a plant cell or plant lacking said first expression cassette.
A method for identifying a compound capable of interacting with a polypeptide comprising a 3′-5′ exonuclease domain comprising:
- a) combining a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:
- 2, SEQ ID NO:
- 4, SEQ ID NO:
- 6, SEQ ID NO:
- 22, SEQ ID NO:
- 10, SEQ ID NO:
- 12, SEQ ID NO:
- 14, SEQ ID NO:
- 16, SEQ ID NO:
- 18, or SEQ ID NO:
- 24 or a homolog thereof, and a compound to be tested for the ability to interact with said polypeptide, under conditions conducive to interaction;
- and
- b) selecting a compound from step (a) that is capable of interacting with said polypeptide.
Dependencies are inferred from the claim text.