Biological nitrogen fixation
AssigneeLand O'Lakes, Inc.
InventorsJames L. Beynon, Frank C. Cannon
Life of this patent
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Abstract
Method for increasing the rate of conversion of atmospheric nitrogen into ammonia in a microorganism of the genus Rhizobium, by increasing the intracellular level of an activator protein which is capable of activating the transcription of DNA of the microorganism encoding one or more proteins capable of effecting such conversion in the microorganism.
Claims19
A method for making a recombinant Rhizobium meliloti microbe having a recombinant gene construct that increases capacity of Rhizobium meliloti to fix nitrogen, and increases biomass of a plant inoculated with the recombinant Rhizobium meliloti, the method comprising:
- making a first plasmid vector having an R. meliloti nifA gene;
- making a second plasmid vector having a gene encoding sequence for a gene promoter selected from the group consisting of B. japonicum nifH activatable gene promoter and B. japonicum nifD activatable gene promoter;
- making a broad host range plasmid vector having a selectable marker gene for transferring the R. meliloti nifA gene and gene promoter to a microbe;
- fusing the R. meliloti nifA gene and the gene sequence encoding for the gene promoter to make an R. meliloti nifA gene::promoter gene construct using the broad host range plasmid vector having a selectable marker gene to position the gene construct on the broad host range plasmid vector;
- transforming the broad host range plasmid vector into an effective host microbe to make a transformed effective host microbe;
- conjugating the transformed effective host microbe with the R. meliloti microbe;
- selecting the R. meliloti transformed by the nifA::promoter gene construct by use of a phenotype expressed by the selectable marker gene;
- and
- inoculating a plant with the selected R. meliloti, thereby increasing the biomass of the plant once the plant matures.
An R. meliloti microbe transformed with a plasmid, the plasmid having a selectable marker gene and a gene construct comprising a gene for an R. meliloti nifA gene and a gene promoter selected from the group consisting of a B. japonicum nifH activatable gene promoter and a B. japonicum nifD activatable gene promoter, the R. meliloti microbe having a phenotype expressed by the selectable marker gene and further having an increased capacity to fix nitrogen and a capacity to increase biomass of a plant inoculated with the R. meliloti.
A method for making a recombinant Rhizobium meliloti microbe having a recombinant gene construct integrated into the R. meliloti microbe's DNA in order to permanently increase the R. meliloti microbe's capacity to fix nitrogen, the method comprising:
- integrating into a symbiotically silent region of Rhizobium meliloti DNA encoding for inositol dehydrogenase (IDH), a cassette comprising:
- a transcriptional promoter gene selected from the group consisting of a B. japonicum nifH activatable gene promoter, and a B. japonicum nifD activatable gene promoter, and fused to an R meliloti nifA gene, to form a construct;
- a selectable marker gene for the selection of integrants carrying at least one cassette;
- effective transcriptional and translational termination signals;
- and
- approximately 3-6 kb of flanking DNA sequences that are homologous to the symbiotically silent region of Rhizobium meliloti DNA encoding for IDH.
A recombinant DNA plasmid having a cassette encoding for genes comprising:
- a transcriptional promoter gene selected from the group consisting of B. japonicum nifH and B. japonicum nifD fused to an R. meliloti nifA gene to form a construct;
- a selectable marker gene for the selection of integrants carrying the cassette;
- transcriptional and translational termination signals to prevent transcriptional read through;
- and
- approximately 3-6 kb of flanking DNA sequences that are homologous to a symbiotically silent region of Rhizobium meliloti DNA encoding for inositol dehydrogenase (IDH).
A recombinant Rhizobium meliloti microbe permanently transformed with an insertion plasmid inserted at a symbiotically silent region of R. meliloti DNA encoding for inositol dehydrogenase (IDH), the insertion plasmid encoding for a cassette comprising:
- a gene promoter selected from the group consisting of B. japonicum nifH and B. japonicum nifD fused to an R. meliloti nifA gene to form a construct;
- a selectable marker gene for the selection of integrants carrying the cassettes;
- transcriptional and translational termination signals to prevent transcriptional read through;
- and
- approximately 3-6 kb of flanking DNA sequences that are homologous to the symbiotically silent region of Rhizobium meliloti DNA encoding for inositol dehydrogenase (IDH).
A method for making a recombinant Bradyrhizobium japonicum microbe having a recombinant gene construct that increases the Bradyrhizobium japonicum microbe's capacity to fix nitrogen, the method comprising:
- making a first plasmid vector having a gene encoding for B. japonicum nifA;
- making a second plasmid vector that includes an R. meliloti fixA gene promoter;
- making a broad host range plasmid vector having a selectable marker gene, the plasmid vector for transferring the B. japonicum nifA gene sequence and the promoter gene sequence to a microbe;
- fusing the Bradyrhizobium japonicum nifA gene sequence and the R. meliloti fixA gene promoter sequence to make a B. japonicum nifA gene::promoter gene construct using the broad host range plasmid vector to position the gene construct on the broad host range plasmid vector;
- transforming the broad host range plasmid vector into an effective host microbe to make a transformed effective host microbe;
- conjugating the transformed effective host microbe with the Bradyrhizobium japonicum microbe to form a transformed host;
- selecting the B. japonicum transformed by the nifA::promoter gene construct by use of a phenotype expressed by the selectable marker gene;
- and
- inoculating a plant with the selected B. japonicum thereby increasing the biomass of the plant once the plant matures.
A Bradyrhizobium japonicum microbe transformed with a plasmid, the plasmid having a selectable marker gene and a recombinant gene construct comprising a gene encoding for Bradyrhizobium japonicum nifA and a gene encoding for an R. meliloti fixA gene promoter, the Bradyrhizobium japonicum microbe having a phenotype expressed by the selectable marker gene and further having an increased capacity to fix nitrogen and a capacity to increase biomass of a plant inoculated with the Bradyrhizobium japonicum.
A method for making a recombinant Bradyrhizobium japonicum microbe having a recombinant gene construct integrated into the Bradyrhizobium japonicum microbe DNA in order to permanently increase the Bradyrhizobium japonicum microbe's capacity to fix nitrogen, the method comprising:
- integrating into a Hind III site of a silent region of Bradyrhizobium japonicum DNA located within a nif gene cluster of Bradyrhizobium japonicum, a cassette comprising:
- a Rhizobium meliloti fixA promoter gene fused to a Bradyrhizobium japonicum nifA gene to form a gene construct;
- a selectable marker gene for the selection of integrants carrying at least one cassette;
- transcriptional and translational termination signals;
- and
- approximately 3-6 kb of flanking DNA sequences that are homologous to the Hind III site of the silent region located within the nif cluster of Bradyrhizobium japonicum DNA.
A Bradyrhizobium japonicum bacterium transformed with an insertion plasmid inserted at a Hind III site within a silent region of a nifgene cluster of Bradyrhizobium japonicum DNA, the insertion plasmid encoding for an R. meliloti fixA gene promoter fused to a nifA gene to form a gene construct;
- DNA sequences homologous to the Hind III site within the silent region of the nif gene cluster of Bradyrhizobium japonicum DNA flanking either side of the gene construct;
- a sequence encoding for a selectable marker gene;
- and effective transcriptional and translational termination signals.
A recombinant DNA plasmid having a cassette encoding for genes comprising:
- an R. meliloti fixA gene promoter fused to a Bradyrhizobium japonicum nifA gene to form a gene construct;
- a selectable marker gene for the selection of integrants carrying at least one cassette;
- transcriptional and translational termination signals to prevent transcriptional read through;
- and
- approximately 3-6 kb of flanking DNA sequences that are homologous to a Hind III site of a silent region of Bradyrhizobium japonicum DNA located within a nif gene cluster of Bradyrhizobium japonicum.
Dependencies are inferred from the claim text.