Methods for obtaining modified phenotypes in plant cells
AssigneeCommonwealth Scientific and Industrial Research Organization (CSIRO)
InventorsPeter Michael Waterhouse, Ming-Bo Wang
Life of this patent
Estimating the term…
Abstract
Methods are provided for reducing the phenotypic expression of a nucleic acid of interest in plant cells, by providing aberrant, preferably unpolyadenylated, target-specific RNA to the nucleus of the host cell. Preferably, the unpolyadenylated target-specific RNA is provided by transcription of a chimeric gene comprising a promoter, a DNA region encoding the target-specific RNA, a self-splicing ribozyme and a DNA region involved in 3′ end formation and polyadenylation.
Claims31
A method for reducing the phenotypic expression of a nucleic acid of interest, which is normally capable of being expressed in a plant cell, said method comprising the steps of
- a) providing to the nucleus of said plant cell unpolyadenylated RNA comprising a target specific nucleotide sequence produced by transcription of a chimeric DNA comprised within said plant cell, said chimeric DNA comprising a plant-expressible promoter operably linked to a target specific DNA region encoding said RNA, said target-specific DNA region comprising a nucleotide sequence of at least 10 consecutive nucleotides having at least about 70% sequence identity to about 100% sequence identity to said nucleic acid of interest and wherein said chimeric DNA farther comprises a DNA region involved in 3′ end formation and polyadenylation, preceded by a self-splicing ribozymne encoding DNA region;
- and
- b) selecting said plant cell wherein the phenotypic expression of said nucleic acid of interest is reduced.
A method for reducing the phenotypic expression of a nucleic acid of interest, which is normally capable of being expressed in a plant cell, said method comprising the steps of
- a) introducing into the nuclear genome of said plant cell a chimeric DNA to generate a transgenic plant cell, said chimeric DNA comprising the following operably linked parts:
- i) a plant-expressible promoter region;
- ii) a target-specific DNA region comprising a nucleotide sequence of at least 10 consecutive nucleotides having at least about 70% sequence identity to about 100% sequence identity to said nucleic acid of interest;
- iii) a DNA region encoding a self-splicing ribozyme;
- and
- iv) a DNA region involved in 3′ end formation and polyadenylation comprising a polyadenylation site
- wherein said chimeric DNA when transcribed produces a first RNA molecule comprising a target specific nucleotide sequence and a self-splicing ribozyme, which when cleaved by autocatalysis produces a second RNA molecule comprising a target specific nucleotide sequence wherein the 3′ end of the first RNA molecule comprising the polyadenylation site has been removed;
- and
- b) isolating a transgenic cell wherein said phenotypic expression of said nucleic acid of interest is reduced.
A chimeric DNA molecule for reducing the phenotypic expression of a nucleic acid of interest, which is normally capable of being expressed in a plant cell, said chimeric DNA molecule comprising
- a) a plant-expressible promoter region;
- b) a target-specific DNA region comprising a nucleotide sequence of at least 10 consecutive nucleotides having at least about 70% sequence identity to about 100% sequence identity to said nucleic acid of interest;
- c) a DNA region encoding a self-splicing ribozyme;
- and
- d) a DNA region involved in 3′ end formation and polyadenylation comprising a polyadenylation site;
- wherein said chimeric DNA when transcribed produces a first RNA molecule comprising a target specific nucleotide sequence and a self-splicing ribozyme, which when cleaved by autocatalysis produces a second RNA molecule comprising a target specific nucleotide sequence wherein the 3′ end of the first RNA molecule comprising the polyadenylation site has been removed.
A method for identifying a phenotype associated with the expression of a nucleic acid of interest in a plant cell, said method comprising:
- a) selecting within said nucleic acid of interest a target sequence of at least 10 consecutive nucleotides;
- b) introducing a chimeric DNA into the nucleus of a suitable plant host cell comprising said nucleic acid of interest, said chimeric DNA comprising the following operably linked DNA fragments:
- i) a plant-expressible promoter region;
- ii) a target-specific DNA region comprising a nucleotide sequence of at least about 70% to about 100% sequence identity to said target sequence;
- followed by
- iii) a DNA region encoding a self-splicing ribozyme located immediately upstream of
- iv) a DNA region involved in 3′ end formation and polyadenylation;
- and
- c) observing the phenotype by a suitable method.
A method for reducing the phenotypic expression of a nucleic acid of interest, which is normally capable of being expressed in cells of a plant, said method comprising the steps of
- a) introducing into the nuclear genome of cells of said plant a chimeric DNA to generate a transgenic plant cell, said chimeric DNA comprising the following operably linked parts:
- i) a plant-expressible promoter region;
- ii) a target-specific DNA region comprising a nucleotide sequence of at least 10 consecutive nucleotides having at least about 70% sequence identity to about 100% identity to said nucleic acid of interest;
- iii) a DNA region encoding a self-splicing ribozyme;
- and
- iv) a DNA region involved in 3′ end formation and polyadenylation comprising a polyadenylation site
- wherein said chimeric DNA when transcribed produces a first RNA molecule comprising a target specific nucleotide sequence and a self-splicing ribozyme, which when cleaved by autocatalysis produces a second RNA molecule comprising a target specific nucleotide sequence wherein the 3′ end of the first RNA molecule comprising the polyadenylation site has been removed;
- b) regenerating a plant from said transgenic cell;
- and
- c) isolating said plant wherein said phenotypic expression of said nucleic acid of interest is reduced.
Dependencies are inferred from the claim text.