NOVEL SELECTABLE MARKER GENES
The subject invention relates to a novel gene referred to herein as DSM-2. This gene was identified in Streptomyces coelicolor A3. The DSM-2 protein is distantly related to PAT and BAR. The subject invention also provides plant-optimized genes encoding DSM-2 proteins. DSM-2 can be used as a transgenic trait to impart tolerance in plants and plant cells to the herbicides glufosinate and bialaphos. One preferred use of the subject genes areas selectable markers. The use of this gene as a selectable marker in a bacterial system can increase efficiency for plant transformations. Use of DSM-2 as the sole selection marker eliminates the need for an additional medicinal antibiotic marker (such as ampicillin resistance) during cloning. Various other uses are also possible according to the subject invention.
1 . A transgenic plant cell comprising a polynucleotide that encodes a protein that has phosphinothricin acetyltransferase activity, wherein said polynucleotide hybridizes under conditions of 6×SSC at 65° C. with the full complement of a nucleic acid probe that encodes SEQ ID NO:2.
2 . The cell of claim 1 wherein said probe comprises a sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:3.
3 . The cell of claim 1 wherein said protein has at least 95% sequence identity with the amino acid sequence of SEQ ID NO:2.
4 . A vector comprising a promoter operable in a plant cell, and a polynucleotide operably linked to said promoter, wherein said polynucleotide encodes a protein that has phosphinothricin acetyltransferase activity, and wherein said polynucleotide hybridizes under conditions of 6×SSC at 65° C. with the full complement of a nucleic acid probe that encodes the amino acid sequence of SEQ ID NO:2.
5 . A method of selecting for a plant cell of claim 1 wherein said method comprises providing a vector of claim 4 to a plurality of plant cells, and growing said plurality of cells in a concentration of a herbicide that permits cells that express said polynucleotide to grow while killing or inhibiting the growth of cells that do not comprise said vector, wherein said herbicide comprises phosphinothricin.
6 . The method of claim 5 wherein said herbicide is selected from the group consisting of bialophos and glufosinate.
7 . The method of claim 5 wherein said method further comprises identifying and regenerating transformed plant cells.
8 . A plant comprising a plurality of cells of claim 1 .
9 . A seed of a plant of claim 8 .
10 . A plant cell of claim 1 wherein said cell further comprises an insect-resistance gene derived from an organism selected from the group consisting of Bacillus thuringiensis, Photorhabdus , and Xenorhabdus.
11 . A plant cell of claim 1 wherein said cell further comprises a second herbicide-resistance gene.
12 . The plant of claim 8 where said plant produces said protein.
13 . The plant of claim 12 wherein said protein comprises SEQ ID NO:2.
14 . A method of using a phosphinothricin-resistance gene as a resistance marker in plant cells, said method comprising the steps of subjecting a plurality of plant cells to transformation with a vector of claim 4 , culturing the cells in a medium, exposing the cells to phosphinothricin, and determining whether cells are resistant to the phosphinothricin.
15 . A process for generating phosphinothricin resistant plant cells, plants, and their propagates wherein said method comprises transforming plant cells with the vector of claim 4 , and regenerating the transformed plant cells to plants that produce propagates.
16 . A process for the production of a phosphinothricin-resistant plant wherein said method comprises incorporating into the genome of the plant a vector of claim 4 , and selecting for phosphinothricin resistance.
17 . A process for the production of a plant cell that is tolerant to the herbicidal activity of a glutamine synthetase inhibitor including phosphinothricin or a compound with a phosphinothricin moiety, wherein said method comprises the step of incorporating into the nuclear genome of a starting plant cell a recombinant DNA comprising: a) a promoter recognized by a polymerase of said starting plant cell, and b) a coding region comprising a DNA fragment from a Streptomyces coelicolor A3 microorganism that produces said glutamine synthetase inhibitor, wherein said DNA fragment encodes a protein with acetyltransferase activity to said glutamine synthetase inhibitor.