# US 2014/0196178 A1 · Plant self nitrogen fixation by mimicking prokaryotic pathways > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2014/0196178 A1 - Document ID: US20140196178A1 - Status: Published application - Kind: A1 - Application number: 14/093,920 - Filed: 2013-12-02 - Published: 2014-07-10 - Earliest claimed: 2012-12-03 - Inventors: Adi Zaltsman - Web page: https://patentgrep.com/patent/US20140196178A1 ## CPC - C12N 15/8243: {involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine} - C12N 15/8261: {with agronomic (input) traits, e.g. crop yield} - Y02A 40/146: Genetically Modified [GMO] plants, e.g. transgenic plants - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE ## Abstract A method for engineering a transgenic plant that replicates the nitrogen fixation mechanism of photosynthetic bacteria, such as cyanobacteria, or that of other bacteria, by targeting to or expressing in plant plastids bacterial nif (nitrogen fixation) genes is provided. A method for reducing the overall concentration of nitrogen in soil is provided using a plant that replicates the nitrogen fixation mechanism of photosynthetic bacteria. Progeny of a plant that replicates the nitrogen fixation mechanism of photosynthetic bacteria is also provided. ## Claims ### Claim 1 A transgenic plant exhibiting enhanced nitrogen fixatation produced by a method comprising the steps of: contacting one or more plant cells with a recombinant nucleic acid sequence encoding Nif-H, Nif-D and Nif-K genes operatively linked to a promoter sequence and a terminator sequence; regenerating one or more plants from the plant cells; and selecting one or more plants, cultivated from the plant cells, exhibiting enhanced nitrogen fixation, wherein said one or more plants comprise the recombinant nucleic acid sequence encoding the Nif-H, Nif-D and Nif-K genes. ### Claim 2 (depends on 1) Seeds, stems, leaves, or roots of the transgenic plant of claim 1, wherein the seeds, stems, leaves or roots comprise the recombinant nucleic acid sequence encoding the Nif-H, Nif-D and Nif-K genes. ### Claim 3 (depends on 1) A transgenic plant as recited in claim 1, wherein the transgenic plant is selected from a group consisting of canola, corn, rice, tobacco, soybean, cotton, alfalfa, tomato, wheat, potato and barley, ### Claim 4 (depends on 3) A transgenic plant as recited in claim 3, wherein the transgenic plant is tomato or tobacco. ### Claim 5 (depends on 1) A progeny of the transgenic plant of claim 1, wherein said progeny comprise the recombinant nucleic acid sequence encoding the Nif-H, Nif-D and Nif-K genes. ### Claim 6 (depends on 3) A progeny of the plant of claim 3, wherein said progeny comprise the recombinant nucleic acid sequence encoding the Nif-H, Nif-D and Nif-K genes. ### Claim 7 (depends on 4) A progeny of the plant of claim 4, wherein said progeny comprise the recombinant nucleic acid sequence encoding the Nif-H, Nif-D and Nif-K genes. ### Claim 8 (depends on 1) A transgenic plant as recited in claim 1, wherein the step of selecting one or more plants comprises growing the one or more plants under conditions of low nitrogen input. ### Claim 9 (depends on 1) A transgenic plant as recited in claim 1, wherein the recombinant nucleic acid sequence encoding the Nif-H, Nif-D and Nif-K genes that contacts the one or more cells is obtained from a source selected from a group consisting of cyanobacteria, azotobacteraceae, rhizobia, and frankia. ### Claim 10 (depends on 1) A method for reducing the overall concentration of nitrogen in soil comprising: placing the transgenic plant of claim 1 in contact with the soil so that as the transgenic plant grows nitrogen is used from the soil thereby reducing the overall concentration of nitrogen in the soil. ### Claim 11 A transgenic plant exhibiting enhanced nitrogen fixatation produced by a method comprising the steps of: contacting a plant cell with a recombinant nucleic acid sequence encoding one of SEQ. ID. NO. 1, SEQ. ID. NO. 2 and SEQ. ID. NO. 3 to produce a transgenic plant; regenerating one or more transgenic plants from the plant cell; and selecting one or more transgenic plants cultivated from the plant cell, wherein the selected transgenic plants each exhibit enhanced nitrogen fixation, wherein the selected one or more transgenic plants each comprise the recombinant nucleic acid sequence encoding SEQ. ID. No. 1, SEQ. ID. No. 2 or SEQ. ID. NO. 3. ### Claim 12 (depends on 11) Seeds, stems, leaves or roots of the transgenic plant of claim 11, wherein the seeds, stems, leaves or roots comprise the recombinant nucleic acid sequence encoding one of SEQ. ID. NO. 1, SEQ. ID. NO. 2 and SEQ. ID. NO. 3. ### Claim 13 (depends on 11) A transgenic plant as recited in claim 11, wherein the transgenic plant is selected from the group consisting of canola, corn, rice, tobacco, soybean, cotton, alfalfa, tomato, wheat, potato and barley. ### Claim 14 (depends on 11) A plant-derived commercial product, which is derived from a transgenic plant produced according to the method of claim 11, wherein said transgenic plant comprises a recombinant nucleic acid sequence encoding one of SEQ. ID. NO. 1, SEQ. ID. NO. 2 and SEQ. ID. NO. 3. ### Claim 15 (depends on 14) A plant-derived commercial product as recited in claim 14, wherein said commercial product is pulp, paper, a paper product, lumber, cigarette, cigar, chewing tobacco, bread, flour, cereal, oat meal, or rice. ### Claim 16 (depends on 11) A method for reducing the overall concentration of nitrogen in soil comprising: placing the transgenic plant of claim 11 in contact with the soil so that as the plant grows nitrogen is used from the soil thereby reducing the overall concentration of nitrogen in the soil. ### Claim 17 A transgenic plant exhibiting enhanced nitrogen fixatation produced by a method comprising the steps of: contacting a plant cell with a recombinant nucleic acid sequence encoding one of SEQ. ID. NO. 1, SEQ. ID. NO. 2 and SEQ. ID. NO. 3 to produce a transgenic plant; contacting the plant cell with at least one synthetic coding DNA sequence (sCDS) optimized for tomato chloroplast expression; regenerating one or more transgenic plants from the plant cell; and selecting one or more transgenic plants, cultivated from the plant cell exhibiting enhanced nitrogen fixation, wherein said one or more plants comprise the recombinant nucleic acid sequence encoding SEQ. ID. NO. 1, SEQ. ID. NO. 2 or SEQ. ID. NO. 3 and the sCDS. ### Claim 18 (depends on 17) A transgenic plant as recited claim 17, wherein the transgenic plant is selected from the group consisting of canola, corn, rice, tobacco, soybean, cotton, alfalfa, tomato, wheat, potato and barley. ### Claim 19 (depends on 17) A transgenic plant as recited in claim 17, wherein the sCDS comprises one of a group consisting essentially of SEQ. ID. NO. 29, SEQ. ID. NO. 30, SEQ. ID. NO. 31, SEQ. ID. NO. 32, SEQ. ID. NO. 33, SEQ. ID. NO. 34, SEQ. ID. NO 35, SEQ. ID. NO. 36, SEQ. ID. NO. 37, SEQ. ID. NO. 38, SEQ. ID. NO. 39 and SEQ. ID. NO. 40. ### Claim 20 (depends on 17) A method for reducing the overall concentration of nitrogen in soil comprising: placing the transgenic plant of claim 17 in contact with the soil so that as the plant grows nitrogen is used from the soil thereby reducing the overall concentration of nitrogen in the soil. ### Claim 21 (depends on 17) A method for increasing nitrogen concentration in soil comprising: placing the transgenic plant of claim 17 in contact with soil so that the plant releases nitrogen into the soil as the plant decays. ### Claim 22 A transgenic plant exhibiting enhanced nitrogen fixatation produced by a method comprising the steps of: contacting a plant cell with a recombinant nucleic acid sequence encoding one of SEQ. ID. NO. 46, SEQ. ID. NO. 47, SEQ. ID. NO. 48 and SEQ. ID. NO. 49 to produce a transgenic plant; regenerating one or more transgenic plants from the plant cell; and selecting one or more transgenic plants cultivated from the plant cell, wherein the selected plants each exhibit enhanced nitrogen fixation, wherein the selected one or more transgenic plants each comprise the recombinant nucleic acid sequence encoding SEQ. ID. NO. 46, SEQ. ID. NO. 47, SEQ. ID. NO. 48 or SEQ. ID. NO. 49. ### Claim 23 (depends on 22) Seeds, stems, leaves or roots of the transgenic plant of claim 22, wherein the seeds, stems, leaves or roots comprise the recombinant nucleic acid sequence encoding one of SEQ. ID. NO. 46, SEQ. ID. NO. 47, SEQ. ID. NO. 48 and SEQ. ID. NO. 49. ### Claim 24 (depends on 22) A transgenic plant as recited in claim 22, wherein the transgenic plant is selected from the group consisting of canola, corn, rice, tobacco, soybean, cotton, alfalfa, tomato, wheat, potato and barley. ### Claim 25 (depends on 22) A transgenic plant-derived commercial product, which is derived from a transgenic plant produced according to the method of claim 22, wherein said transgenic plant comprises a recombinant nucleic acid sequence encoding one of SEQ. ID. NO. 46, SEQ. ID. NO. 47, SEQ. ID. NO. 48 and SEQ. ID. NO. 49. ### Claim 26 (depends on 25) A transgenic plant-derived commercial product as recited in claim 25, wherein said commercial product is pulp, paper, a paper product, lumber, cigarette, cigar, chewing tobacco, bread, flour, cereal, oat meal, or rice. ### Claim 27 (depends on 22) A method for reducing the overall concentration of nitrogen in soil comprising: placing the transgenic plant of claim 22 in contact with the soil so that as the plant grows nitrogen is used from the soil thereby reducing the overall concentration of nitrogen in the soil. ### Claim 28 A transgenic plant exhibiting enhanced nitrogen fixatation produced by a method comprising the steps of: contacting one or more transgenic plant cells with a recombinant nucleic acid sequence encoding a Nif-H gene operatively linked to a first promoter; contacting the transgenic plant cells with a recombinant nucleic acid sequence encoding a Nif-D gene operatively linked to a second promoter; contacting the transgenic plant cells with a recombinant nucleic acid sequence encoding a Nif-K gene operatively linked to a third promoter; regenerating one or more transgenic plants from the plant cells; and selecting one or more transgenic plants cultivated from the plant cells, wherein the selected transgenic plants each exhibit enhanced nitrogen fixation, wherein the selected transgenic plants each comprise the recombinant nucleic acid sequence encoding the Nif-H gene, the recombinant nucleic acid sequence encoding the Nif-D gene and the recombinant nucleic acid sequence encoding the Nif-K gene. ### Claim 29 (depends on 28) A transgenic plant as recited in claim 28, wherein the recombinant nucleic acid sequence encoding the Nif-H gene is selected from a group consisting essentially of SEQ. ID. NOs. 29, 32, 35 and 38. ### Claim 30 (depends on 28) A transgenic plant as recited in claim 28, wherein the recombinant nucleic acid sequence encoding the Nif-D gene is selected from a group consisting essentially of SEQ. ID. NOs. 30, 33, 36 and 39. ### Claim 31 (depends on 28) A transgenic plant as recited in claim 28, wherein the recombinant nucleic acid sequence encoding the Nif-K gene is selected from a group consisting essentially of SEQ. ID. NOs. 31, 34, 37 and 40. ### Claim 32 (depends on 28) A transgenic plant as recited in claim 28, wherein the first promoter is selected from a group consisting essentially of SEQ. ID. NOs. 41-45. ### Claim 33 (depends on 28) A transgenic plant as recited in claim 28, wherein the second promoter is selected from a group consisting essentially of SEQ. ID. NOs. 41-45. ### Claim 34 (depends on 28) A transgenic plant as recited in claim 28, wherein the third promoter is selected from a group consisting essentially of SEQ. ID. NOs. 41-45. ### Claim 35 (depends on 28) A transgenic plant as recited in claim 28, wherein at least one of the promoters comprises SEQ. ID. NO. 4 or SEQ. ID. NO. 28.