TH1801000267A - Methods and compositions for improving plant reproductive characteristics. - Google Patents

Abstract

26/03/2018(OCR)

Claims (1)

11/12/2018(OCR) Page 1 of 13 Claims 1. A method for increasing nitrogen fixation in non-legume plants, comprising: exposing the plant to multiple bacteria, each member of the multiple bacteria. That includes one or more genetic variations inserted in one or more genes or non-coding polynucleotides in the bacterial nitrogenase or genetic control network of a bacterium. Scimitation, such that bacteria can extract nitrogen from the atmosphere in the presence of exogenous nitrogen, where the bacteria are not microorganisms within a different genus, and where bacteria in plants produce 1% or more of the nitrogen absorbed in Plant 2. The method of Corollary 1, wherein one or more genetic variations comprising an inserted control sequence operably linked to one or more such genes in Direct nitrogen or assimylation genetic control network 3. The method of claim 2, wherein the control sequence is a promoter 4. The method of claim 3, wherein The promoter is an inducible promoter. 5. The method of claim 1, wherein the bacterium does not comprise a uniformly expressed promoter operably linked to a nitrogen directive gene or a genetic regulatory network of Assimylation 6. The method of claim 1, wherein the bacterium does not comprise a uniformly expressed promoter operably linked to genes in the nif7 gene family. The method of claim. Claim 1, wherein the bacteria in the plant excrete nitrogen-containing products of nitrogen fixation 8. The method of claim 1, wherein the bacteria that the ducks infect with the plant do not stimulate an increase in nitrogen uptake from 9. The method of claim 1, wherein the plant is grown in soil from a field which has been supplied with at least 50 pounds of nitrogen-containing fertilizer per acre, and wherein the nitrogen-containing fertilizer is including at least 5% nitrogen by weight. 10. The method of claim 9, wherein the nitrogen containing fertilizer comprises ammonium or ammonium-containing molecules, page 2 of number 13, page 11. The method of claim 1, wherein the bacteria comprising at least two different species of bacteria. 12. The method of claim 1, wherein the bacteria comprising at least two different species of bacteria. with at least two different strains of the same genus of bacteria. 13. The method of claim 1, wherein the exogenous nitrogen is selected from a fluff comprising one or more of glutamine, ammonia, ammonium, urea, nitrate, Nitrite, ammonium-containing molecule, nitrate-containing molecule, and nitrite-containing molecule 14. The method of claim 1, wherein the plant bacteria produce 5% or more of nitrogen. that is right in the plant 15. The method of claim 1, wherein the bacteria in the plant produce 10% or more of the direct nitrogen in the plant. 16. The method of claim 1, wherein one gene is More than likely, nucleotides not encoded in the bacterium's nitrogen ligation or ascimylation genetic control networks were selected from groups that included: nifA,a// L,ntrB,ntrC,glutamine synthetase encoding polynucleotide1,glnA,glnB,glnK,drat1amtB,glutamine synthetase encoding polynucleotide1. ,glnD,glnE,ก//ก,ก//H,ก//ท),ก/'/K,ก//Y,ก//ท,ก//N,ก/'/บ,nilr ,ก//V,ป//ป,ก/TZ,ก/'/M,ก//Th,ก//R,andก//Q17.The method of claim 11, wherein the variation One or more genetic mutations that result in one or more of: increased expression or activity of NifA glutaminase increased expression or activity of NifA Decrease of NifL, NtrB, Glutamine Synthetase, GlnB, GlnK, DraT, AmtB have a reduced adenylyl scavenging effect of GlnE or a reduced uracyl scavenging effect of GlnD18. Method of Claim 1, where one or more genetic variations are (A) knock-out mutations, (B) alter or destroy the regulatory sequence of the target gene, or (C) consist of insertions. of different control sequences 19. The method of claim 1, wherein the bacteria belong to the genus comprising Enterobacter, Ranella, Gosagopia, Bercolder or Kelp. Siella 20. The method of claim 1, wherein the bacteria are endophytic, epiphytic, or rhizomatous. Page 3 of 13 21. The method of claim 1. Claim 1, wherein the bacteria are present in at least the plant roots such that the bacteria are present in the plant in an amount of at least approximately 105 CFU per gram of new weight of tissue. 22. The method of claim. Right number 1, where the plant is an agricultural crop 23. The method of claim 22, wherein the agricultural crops are selected from rice straw, canola, tomato, strawberry, barley, rice, corn meze, and wheat. 24. Method. The method of claim 22, wherein the plant is a genetically modified organism. 25. The method of claim 22, wherein the plant is not a genetically modified organism. 26. The method of claim 22, wherein the plant is a genetically modified organism. 22, where plants are genetically engineered or genetically modified for efficient nitrogen use. 27. A bacterial population consisting of bacteria containing one or more genetic variations inserted in one gene or more than several non-coding nucleotides. In the nitrogen deposition of bacteria or the genetic control network of assylation, such that bacteria can extract nitrogen from the atmosphere in the presence of external nitrogen without Bacteria are microorganisms within different genera, and bacteria in plants produce 1% or more of the nitrogen required in growing plants in the presence of a bacterial population. 28. The bacterial population of a plant is: Claim 27, wherein one or more genetic variations comprising an inserted control sequence operably linked to one or more genes in the nitrogen channel or regulatory network. genetics of aschemylation 29. The bacterial population of claim 28, wherein the control sequence is a promoter. 30. The bacterial population of claim 29, wherein the promoter is an inducible promoter. 31. The bacterial population of claim 27, provided that the bacterium does not include a uniformly expressed promoter that is operatively linked to the genes for nitrogen metabolism or the genetic control network of aschemylation. 32. The bacterial population of claim 27, wherein the bacteria do not comprise a uniformly expressed promoter that is functionally linked to genes in the nif gene family. 33. The bacterial population of claim 27. 27, where bacteria in plants excrete nitrogen-containing products of excretion, page 4 of 13, page 34. The bacterial population of claim 27, where the bacteria exposed to the plant do not stimulate an increase in nitrogen uptake from outside the atmosphere other than from the atmosphere 35. The bacterial population of claim 27, wherein the bacterial population consists of at least two thousand different families of bacteria. 36. The bacterial population of claim 27, wherein the bacterial population consists of at least two different strains of the same species of bacteria. 37. The bacterial population of claim 27, where external nitrogen is selected from a fertilizer consisting of one or more of glutamine, ammonia, ammonium, urea, nitrate, nitrite, ammonium-containing molecules, The first nitrate, and the nitrite molecule. 38. The bacterial population of claim 27, wherein the bacteria in the plant produce 5% or more of the nitrogen that is direct in the plant. 39. The bacterial population of claim 27, wherein the bacteria in the plant produce 10% or more of the nitrogen that is direct in the plant. 40. The bacterial population of claim 27, where one gene or more polynuclei is present. Non-coding peptides of the bacterial nitrogen directive or assimylation genetic regulatory network were selected from groups consisting of: nifA, nilL, ก?/A, ntrC, glutamine. polynucleotide-encoded natase,gink,glnB,th/kK,drat,amtB,polynucleotide-encoded suitamine,th/kor,th /กE,ก//ช,nitH,ก//A,nitK,ก//Y,ก//E,nitN,ก//B,ก//R,ก//'V,ก//P ,a//z,nitM,a//F,a//th,anda//Q41.The bacterial population of claim 27, wherein one genetic variation Or rather, it is a mutation that results in one or more of: increased expression or activity of NifA, glutamine, decreased expression or activity of NifL, NtrB. ,Glutamine synthetel,GlnB,GlnK,DraT,AmtB,decreased adenysyl-elimination effect of GlnE or,decreased urodysyl-elimination effect of GlnD42.The bacterial population of claim 27, wherein one genetic variation Or is it more like (A) knock-out mutation? (B) altering or destroying the regulatory sequence of the target gene; (C) comprising inserting a heterologous regulatory sequence; 43. The bacterial population of claim 27, wherein the multiple bacteria belong to the genus comprising with Enterobacter raphela, Gosagonia, Bercolder or Klepsiella, page 5 of 13, page 44. The bacterial population of claim 27, wherein The bacteria are endophydic, epiphydic, or rhizostitial. 45. The bacterial population of claim 27, where the plant is an agricultural crop. 46. The bacterial population of claim 2. 45, wherein the agricultural crops are selected from sorghum, canola, tomato, strawberry, barley, rice, mesh corn, and wheat; 47. The bacterial population of claim 45, wherein The plant is a genetically modified organism. 48. The bacterial population of claim 45, wherein the plant is not a genetically modified organism. 49. The bacterial population of claim 45, wherein the plant is genetically engineered or modified. Varieties for efficient nitrogen use 50. The bacterial population of claim 27, wherein several bacteria occupy the plant such that bacteria are present in the plant in an amount of at least approximately 106 cfu per gram of new weight of the plant. 51. Composition. comprising the bacterial population of claim 2750 any one of 52. Composition of claim 51, wherein the composition includes the population Bacterial coating on the seed surface. 53. Composition of claim 51, wherein the composition is formulated as a liquid or powder. 54. The separated bacteria accumulated are ATCCaccessionDepositNo.PTA122293 or PTA122294. 55. Non-genus bacteria that contain one or more genetic variations inserted into one gene or more non-coding nucleotides of the bacterial nitrogen signature? The genetic control network of aschemylation, such that bacteria are able to obtain nitrogen from the atmosphere in the presence of exogenous nitrogen56. The bacteria of claim 55, wherein one genetic variation or More than comprising an inserted control sequence operably linked to one or more such genes of a nitrogen binding or ascemylation genetic control network 57. The bacteria of claim no. 56, wherein the control sequence is a promoter. 58. The bacteria of claim 57, wherein the promoter is an inducible promoter page 6 of 13. 59. The bacteria of claim 55, wherein the bacteria are not, comprising a regularly expressed promoter that is operatively linked to the nitrogen directive gene or the ascimylation genetic regulatory network 60. The bacterium of claim 55. ,wherein the bacterium does not ,comprise a promoter that Consistently expressed expression that is operatively linked to genes in the bacterial nif61 gene family of claim 55, wherein one or more genes or nucleotides do not encode in The bacterial nitrogen or assimilation genetic control network was selected from a group consisting of: A/TA, nitL, ntrB, ntrC, glutamine synthetase encoding. linucleotide, ก/กA, ginB, glnK, drat, amtB, Sutaminase that encodes posinucleotide, ก/กD, ก/กE, ก/ /P,ก//ก,nif, nifK,nifY,ก/E,ก/'/N,nif,ก,ก//ก,ก//V1ก//ป,ก//Z,ก/7M ,A/E,A/?,andA//Q62.The bacteria of claim 55, wherein one or more genetic variations are mutations that result in one or more of:expressions. or increased activity of NifA and glutamine expression or decreased activity of NifL, NtrB, glutamine synthetase, GlnB, GlnK, DraT ,AmtB acts on the reduced adenylene clearance of GlnE? The reduced chelating activity of GlnD63 bacteria of claim 55, wherein one or more of the genetic variations is (A) a knockout mutation (B (C) altering or destroying the regulatory sequence of the target gene; (C) comprising inserting a heterologous regulatory sequence; 64. The bacterium of claim 55, wherein the bacterium is from the genus comprising Pterobacter, Ranella, Gosagopia, Bercolder or Klepsiella 65. The bacteria of claim 55, wherein the bacteria are endophytic, Epiphytic, or raichos pericarp66. A method for producing one or more bacteria, comprising: (l) isolating the bacteria from the tissue or soil of a plant. (g) Inserting genetic variation into one or more bacteria to produce one or more variable bacteria. (c) Multiplying plants with variable bacteria. Page 7 of 13. (d) Separation of bacteria from tissue. or soil of one plant, where the plant from which the bacterium has been isolated has improved genetic characteristics relative to the other plants, and (e) the cuttings (g) to (d). With the bacteria isolated in step (d) 67. The method of claim 66, wherein the improved genotype is triangulated. Nitrogen is enriched in plants from which bacteria are isolated. 68. The method of claim 66, wherein the genetic variation is variation in genes selected from the group consisting of: nifA, nifL, ntrB, ntrC,glnA,glnB,glnK,draT,amtB,glnD,glnE,nifj,nifH,nifD,nifK1nifY,nifE,nifN,nifu,nifS,nifV,nifw,nifZ,nifM,nifF,nifB,andnifQ69.Method of Claim 66, where genetic variation is variation in genes that Encodes a protein with the presence of functional groups selected from the group that includes: glutamine synthetase, glutamine, glutamine synthetase adenylyltrans. Spherol, Transcription Activator, Anti-Transcription Activator, Pyruvate Flavodoxin Oxidoretactel, Flavodoxin, or NAD+W Nitrogen Retaktel. ADPD ribosyltransferase test 70. The method of claim 66, wherein the genetic variation is a mutation that This results in one or more of: increased expression or activity of NifA or aglutamine, decreased expression or activity of NifL, NtrB, glutamine synthetase, GlnB, GlnK, DraT, AmtB reduce the adenylene cleavage effect of GlnE or the urethral cleavage effect of GlnE. reduction of GlnD71. The method of claim 66, wherein the genetic variation is a knockout mutation. 72. The method of claim 66, wherein the genetic variation results in Removal or destruction of the protein domain 73 occurs. The method of claim 66, wherein the genetic variation alters or destroys the regulatory sequence of the target gene. 74. The method of claim 66, wherein the genetic variation comprises the insertion of a heterogeneous control liver vessel. Page 8 of 13 75. The method of claim 66, wherein wherein the genetic variation comprises the insertion of a regulatory sequence found within the genome of the bacterial species or genus corresponding to the bacterium in which the genetic variation is inserted. 76. The method of claim 75, wherein The control sequence is selected based on the level. Gene expression in the culture of bacterial feathers or within plant tissue77. The method of claim 66, wherein the genetic variation is produced by chemical mutagenesis78. The method of claim 66, wherein step (c) further comprises exposing the plant to a living or inanimate stressor. 79. The method of claim 67, wherein the bacteria isolated after repeating step (g) to (g) one or more times produce 1% or more nitrogen than in the experimental plant with the repeat. slightly identical to the first plant. 80. The method of claim 67, wherein the bacteria separated after treatment, repeating steps (g) to (g) once or more show at least an increase in 2 times the nitrogen retention compared to bacteria isolated from first plants. 81. The method of claim 80, wherein the second plant is grown in the presence of fluff supplemented with glutamine, ammonia, or any other chemical source of nitrogen. 82. The method of claim 80. Claim 66, wherein the first crop is an agricultural crop. 83. The method of claim 82, wherein the agricultural crop is selected from barley, rice, maize, wheat, rice. Straw, sweet corn, sugarcane, onion, tomato, strawberry, or asparagus. 84. The method of claim 66, wherein one plant or plant among several plants is a model plant. 85. The method. The method of claim 84, wherein the model plants are selected from Cheetary, Brachipodium, or Arabidopsis 86. The method of claim 66, wherein the variation Genetic is a genetic variation. Pre-determined genetic variation that is specifically inserted into the target locus 87. The method of claim 66, wherein the genetic variation is a static mutation within the target locus. Page 9 of 13 Page 88 The method of claim 66, wherein step (l) further comprises performing a genetic analysis of the bacterial isolate 89. The method of claim 66, wherein Step (g) further includes applying selective pressure to optimize the bacterium comprising genetic variation. 90. The method of claim 89, wherein the selection pressure comprises segregating a genome lacking genetic variation inserted into a target locus, wherein the segregation occurs within 100 nu. Kleotide of target position 91. The method of claim 89, further comprising isolating bacteria that survive the selection pressure. 92. The method of claim 91, wherein the cleavage is guided by a site-specific nuclease selected from a group consisting of a counterselectable nuclease, ZincFinger nuclease, Ni. CRISPR nuclease, TALE nuclease, or meganuclease 93. Method of claim 92, wherein the site-specific nuclease is CRISPR nuclease 94. Method. As to claim 66, where the genetic variation is inserted? Missing of one nucleotide or more than 95. The method of claim 66, wherein the bacterium is separated after performing steps (g) to (d) once. or rather endophytic, epiphytic, bacterial pathogen 96. The method of claim 66, wherein the bacteria are separated after performing 1 cutting step ( g) to (d) once or more comprising several different taxonomic units of bacterium. 97. The method of claim 66, wherein the bacteria are separated from the plant tissue. 98. The method of claim 66, wherein the bacteria are separated in step (l )comprising the separation of bacterium from the seeds of a plant99. A method for modifying the genome of the shell of a bacterium species, the method comprising: bacterium, wherein the subsequences include a first originating sequence, a promoter, and a second originating sequence in a 5' to 3' direction and (g) induction of the same originating kyombination between the loci. Genome targets and polynucleotides to produce sequences generated by kiombination, where 0). The target position includes the sequence on 10 faces of 13 flanking target faces. by the same first and second gene sequence, and (and) causing the expression of one or more second genes attached to the target location to increase by 100. The method of claim 99, further comprising with:(b) (iii)The homologous combination prevents the expression of a first gene comprising Target sequence 101. The method of claim 99, further comprising: (c) separating the bacterial shell comprising the sequence generated by combination 102. The method of claim 102. The method of claim 101. at 99, where promoters are selected on the basis of their expression being above a threshold level under environmental conditions. 103. The method of claim 102, wherein the environmental conditions are selected from a group consisting of: nutrient availability, nutrient deficiency, nitrogen stress, increased nitrogen exposure, Heat, cold, osmotic stress, drought, flooding, salinity, presence or absence of interspecies transmitting compounds, presence or absence. of pathogenic microorganisms, and the presence or absence of drutile and plant killers, herbicides, insecticides, roundworms, fungicides, or bactericides, wherein the exposure increases or decreases relative to the reference condition. 104. The method of claim 103, wherein the promoter selection comprises: (a) exposure of the shell of the bacterial species to the condition environment, (b) measuring the exposure levels of cellular transcripts, and (c) identifying promoters that drive higher levels of transcript expression. Starting threshold level 105. The method of claim 99, wherein the subsequence does not encode a protein of the bacterial species. 106. The method of claim 99, wherein the subsequence does not encode a specific identifier. Select 107. The method of claim 101, wherein separating the bacterial shell comprises introducing negative selection on cells lacking the sequence generated by recombination. 108. The method of claim 108. Rights 107, wherein negative selection comprises segregation of a genome lacking a sequence generated by 'recombination, where segregation occurs within 100 nucleotides of the target sequence. Page 11 of 13 Page 109. The method of claim 108, wherein the cleavage is guided by a site-specific nuclease selected from a pool consisting of a counter-tabbling agent, ZincFinger nuclease, CRISPR nuclease, TALE nuclease, or meganuclease 110. The method of claim 109, wherein the site-specific nuclease is CRISPR nuclease 111. The method of claim 99, wherein the first gene is a negative regulator of one or more second genes. 112. The method of claim 99, wherein the gene One, one or more genes, or both, are members of the nitrogen direct pathway. 113. The method of claim 99, wherein the first gene is selected from a group consisting of: NifL, NtrB, glutamine synthetel, GlnB, GlnK, DraT, and AmtB114. Claim 99, wherein one or more second genes are selected from a group consisting of: NifA and glutamine 115. The method of claim 99, wherein the bacterial cells The separated bacteriophage is endophytic, epiphytic, or rhizosphere. 116. The method of claim 115, wherein the separated bacterial cells produce 5% or more of the nitrogen in Cells of the host plant 117. The method of claim 116, wherein the isolated bacteria produce nitrogen in the presence of glutamine, ammonia, or Other chemical sources of nitrogen 118. Polynucleotides for blocking target genes in cells of bacterial species, polynucleotides comprising subdivisions containing sequences derived from the species. bacterium, where: (l) the suborder includes a first order of the same origin, a promoter, and a second order of the same origin in a 5' to 3' direction; (b) the first and second order of the same origin. Try to align with the wings that flank the target. is missing when there is a recombinant homolog between the digestive tract and the target site comprising the target sequence. (c) The target sequence includes at least the first protein-coding portion of the target gene. and page 12 of page 13 (d), the subsequence does not encode a protein of bacterial species 119. The polynucleotide of claim 118, wherein the subsequence does not encode a helper. Optional 120. The polynucleotide of claim 118, wherein the promoter is the promoter of a gene whose expression activity is above the reactivation threshold under environmental conditions. 121. The nucleotide of claim 120, wherein the environmental conditions are the increased exposure to nitrogen, and where the increased exposure is relative to the reference conditions 122. The polynucleotide of claim 118, wherein the target sequence is adjacent to one or more second genes. 123. The polynucleotide of claim 122, wherein that the first gene, one or more of the second genes, or both are members of the nitrogen direct pathway 124. The polynucleotide of claim 118, wherein the genes are Selected from the group consisting of: NifL, NtrB, glutamine synthetase, GlnB, GlnK, DraT, and AmtB125. The polynucleotide of claim 118, wherein the shell is a bacterium. which is endophytic, epi1phytic, or rhichosteraceae 126. The expression vector DNA comprising the polynucleotide of claim 118125. 127. The genetically engineered shell comprising the expression vector DNA. expressionvector) of claim 126128. The method of claim 1, wherein several bacteria belong to the genus Enterobacter. 129. The method of claim 11, wherein several of the bacteria belong to the genus Ranella. 130. The method of claim 11, wherein the several bacteria belong to the genus Gosa. The method of claim 1, wherein the bacteria belong to the genus Berkolderei 132. The method of claim 1, wherein the bacteria belong to the genus Klepsiella 133. The bacterial population of claim 27, wherein several bacteria belong to the genus Enter. Robacter 134. The bacterial population of claim 27, wherein several bacteria belong to the genus Ranella. 135. The bacterial population of claim 27, wherein several bacteria belong to the genus Gosatopia. 136. . The bacterial population of claim 27, wherein "several bacteria belong to the genus Burkolderei" page 13 of 13 page 137. The bacterial population of claim 27, wherein "several bacteria belong to the genus" Clepsiella 138. The bacteria of claim 55, wherein the bacteria are from the genus Enterobacter. 139. The bacteria of claim 55, wherein the bacteria are from the genus Ranel. La 140. The bacteria of claim 55, wherein the bacteria are from the genus Gosagonia. 141. The bacteria of claim 55, wherein the bacteria are from the genus Berkolderei. 142. The bacteria of claim 55, wherein the bacteria are from the genus Klepsiella. 143. The method of claim 99, further comprising: (b)(iii) homologous combinations blocking expression of a first gene comprising the target sequence and ( c) Isolation of bacterial shells comprising sequences generated by combination.