constructs

Abstract

The present disclosure relates to promoters, elements associated with promoters, promoter-reporter pairs, and constructs comprising the above elements suitable for plant transformation.

Description

[0001] Cross-references to related applications This application claims priority to U.S. Provisional Application No. 63 / 600,188, filed November 17, 2023, pursuant to 35 USC § 119(e). The contents of that application are incorporated herein by reference in their entirety.

[0002] sequence list This application includes a sequence list, which has been submitted in XML format and is incorporated herein by reference in its entirety. The sequence list was created on November 11, 2024, named 16195_0016-00304_SL.xml, and has a size of 157,582 bytes.

[0003] background In genetically engineered plants, foreign transgenes are introduced into host plants via various constructs to express the transgenes in those host plant cells. However, due to the interactions between different construct elements and the expression mechanisms of host plant cells, combinations of specific regulatory elements with foreign transgenes may not produce the desired transgene expression levels in host plant cells. With the increasing prevalence of genetic engineering of plant cells, there is a need for constructs that can achieve high levels of transgene expression in transformed plant cells. This disclosure addresses this need.

[0004] Overview In some embodiments, this disclosure provides a nucleic acid molecule comprising: a) a promoter comprising a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with a nucleic acid sequence selected from SEQ ID NO: 1-42; and b) a reporter gene, wherein the promoter is operatively linked to the reporter gene such that, in response to exposure to a stressor, the promoter induces expression of the reporter gene in a plant. In some embodiments, the nucleic acid molecule further comprises a 5' UTR comprising a nucleic acid sequence selected from SEQ ID NO: 43-79, wherein the nucleic acid sequence encoding the 5' UTR is operatively linked to a nucleic acid sequence encoding the promoter. In some embodiments, the reporter gene is selected from genes encoding fluorescent proteins, genes encoding bioluminescent proteins, and genes encoding pigment proteins. In some embodiments, the reporter gene encodes a fluorescent protein. In some embodiments, the reporter gene encodes bFLO_GFP. In some embodiments, the nucleic acid molecule is used in soybean (soybean, Glycine max Gene regulatory activity is exhibited in plants.

[0005] In some embodiments, the nucleic acid molecule includes a promoter comprising a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 4. In some embodiments, the promoter is operatively linked to a 5' UTR comprising a nucleic acid sequence comprising SEQ ID NO: 46. In some embodiments, the promoter comprises SEQ ID NO: 4. In some embodiments, the promoter comprises SEQ ID NO: 4, and the 5' UTR comprises SEQ ID NO: 46. In some embodiments, the nucleic acid molecule includes a promoter comprising a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 14. In some embodiments, the promoter is operatively linked to a 5' UTR comprising a nucleic acid sequence comprising SEQ ID NO: 56. In some embodiments, the promoter comprises SEQ ID NO: 14. In some embodiments, the promoter comprises SEQ ID NO: 14, and the 5' UTR comprises SEQ ID NO: 56. In some embodiments, the nucleic acid molecule comprises a promoter that comprises a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 15. In some embodiments, the promoter is operatively linked to a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 57. In some embodiments, the promoter comprises SEQ ID NO: 15. In some embodiments, the promoter comprises SEQ ID NO: 15, and the 5' UTR comprises SEQ ID NO: 57. In some embodiments, the nucleic acid molecule comprises a promoter that comprises a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 16. In some embodiments, the promoter is operatively linked to a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 58. In some embodiments, the promoter comprises SEQ ID NO: 16. In some embodiments, the promoter comprises SEQ ID NO: 16, and the 5' UTR comprises SEQ ID NO: 58. In some embodiments, the nucleic acid molecule comprises a promoter that comprises a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 17. In some embodiments, the promoter is operatively linked to a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 59.In some embodiments, the promoter comprises SEQ ID NO: 17. In some embodiments, the promoter comprises SEQ ID NO: 17, and the 5' UTR comprises SEQ ID NO: 59. In some embodiments, the nucleic acid molecule comprises a promoter that comprises a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 18. In some embodiments, the promoter is operatively linked to a 5' UTR comprising a nucleic acid sequence comprising SEQ ID NO: 60. In some embodiments, the promoter comprises SEQ ID NO: 18. In some embodiments, the promoter comprises SEQ ID NO: 18, and the 5' UTR comprises SEQ ID NO: 60. In some embodiments, the nucleic acid molecule comprises a promoter that comprises a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 19. In some embodiments, the promoter is operatively linked to a 5' UTR containing the nucleic acid sequence of SEQ ID NO: 61. In some embodiments, the promoter contains SEQ ID NO: 19. In some embodiments, the promoter contains SEQ ID NO: 19, and the 5' UTR contains SEQ ID NO: 61. In some embodiments, the nucleic acid molecule contains a promoter that contains a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 1. In some embodiments, the promoter is operatively linked to a 5' UTR containing the nucleic acid sequence of SEQ ID NO: 43. In some embodiments, the promoter contains SEQ ID NO: 1. In some embodiments, the promoter contains SEQ ID NO: 1, and the 5' UTR contains SEQ ID NO: 43. In some embodiments, the nucleic acid molecule includes a promoter comprising a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 20. In some embodiments, the promoter is operatively linked to a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 62. In some embodiments, the promoter comprises SEQ ID NO: 20. In some embodiments, the promoter comprises SEQ ID NO: 20, and the 5' UTR comprises SEQ ID NO: 62.In some embodiments, the nucleic acid molecule includes a promoter comprising a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 21. In some embodiments, the promoter is operatively linked to a 5' UTR comprising a nucleic acid sequence comprising SEQ ID NO: 63. In some embodiments, the promoter comprises SEQ ID NO: 21. In some embodiments, the promoter comprises SEQ ID NO: 21, and the 5' UTR comprises SEQ ID NO: 63. In some embodiments, the nucleic acid molecule includes a promoter comprising a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 22. In some embodiments, the promoter is operatively linked to a 5' UTR comprising a nucleic acid sequence comprising SEQ ID NO: 64. In some embodiments, the promoter comprises SEQ ID NO: 22. In some embodiments, the promoter comprises SEQ ID NO: 22, and the 5' UTR comprises SEQ ID NO: 64. In some embodiments, the nucleic acid molecule comprises a promoter that comprises a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 23. In some embodiments, the promoter is operatively linked to a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 65. In some embodiments, the promoter comprises SEQ ID NO: 23. In some embodiments, the promoter comprises SEQ ID NO: 23, and the 5' UTR comprises SEQ ID NO: 65. In some embodiments, the nucleic acid molecule comprises a promoter that comprises a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 6. In some embodiments, the promoter is operatively linked to a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 48. In some embodiments, the promoter comprises SEQ ID NO: 6. In some embodiments, the promoter comprises SEQ ID NO: 6, and the 5' UTR comprises SEQ ID NO: 48. In some embodiments, the nucleic acid molecule comprises a promoter that comprises a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 24. In some embodiments, the promoter is operatively linked to a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 66. In some embodiments, the promoter comprises SEQ ID NO: 24.In some implementations, the promoter contains SEQ ID NO: 24, and the 5' UTR contains SEQ ID NO: 66.

[0006] In some embodiments, the reporter gene is selected from genes encoding fluorescent proteins, genes encoding bioluminescent proteins, and genes encoding pigment proteins. In some embodiments, the reporter gene encodes a fluorescent protein. In some embodiments, the reporter gene encodes bFLO_GFP. In some embodiments, the nucleic acid molecule is found in soybean (soybean, Glycine max Gene regulatory activity is exhibited in plants.

[0007] In some embodiments, this disclosure provides a binary vector comprising: a) a promoter comprising a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with a nucleic acid sequence selected from SEQ ID NO: 1-42; and b) a reporter gene, wherein the promoter is operatively linked to the reporter gene such that, in response to exposure to a stressor, the promoter induces expression of the reporter gene in a plant. In some embodiments, the binary vector further comprises a 5' UTR comprising a nucleic acid sequence selected from SEQ ID NO: 43-79, wherein the nucleic acid sequence encoding the 5' UTR is operatively linked to a nucleic acid sequence encoding the promoter. In some embodiments, the reporter gene is selected from genes encoding fluorescent proteins, genes encoding bioluminescent proteins, and genes encoding pigment proteins. In some embodiments, the reporter gene encodes a fluorescent protein. In some embodiments, the reporter gene encodes bFLO_GFP. In some embodiments, the binary vector is used in soybean (soybean, Glycine max Gene regulatory activity is exhibited in plants.

[0008] In some embodiments, the binary vector comprises a promoter comprising a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 4. In some embodiments, the promoter is operatively linked to a 5' UTR comprising a nucleic acid sequence comprising SEQ ID NO: 46. In some embodiments, the promoter comprises SEQ ID NO: 4. In some embodiments, the promoter comprises SEQ ID NO: 4, and the 5' UTR comprises SEQ ID NO: 46. In some embodiments, the binary vector comprises a promoter comprising a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 14. In some embodiments, the promoter is operatively linked to a 5' UTR comprising a nucleic acid sequence comprising SEQ ID NO: 56. In some embodiments, the promoter comprises SEQ ID NO: 14. In some embodiments, the promoter comprises SEQ ID NO: 14, and the 5' UTR comprises SEQ ID NO: 56. In some embodiments, the binary vector comprises a promoter containing a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 15. In some embodiments, the promoter is operatively linked to a 5' UTR containing the nucleic acid sequence of SEQ ID NO: 57. In some embodiments, the promoter comprises SEQ ID NO: 15. In some embodiments, the promoter comprises SEQ ID NO: 15, and the 5' UTR comprises SEQ ID NO: 57.

[0009] In some embodiments, the binary vector comprises a promoter comprising a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 16. In some embodiments, the promoter is operatively linked to a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 58. In some embodiments, the promoter comprises SEQ ID NO: 16. In some embodiments, the promoter comprises SEQ ID NO: 16, and the 5' UTR comprises SEQ ID NO: 58. In some embodiments, the binary vector comprises a promoter comprising a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 17. In some embodiments, the promoter is operatively linked to a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 59. In some embodiments, the promoter comprises SEQ ID NO: 17. In some embodiments, the promoter comprises SEQ ID NO: 17, and the 5' UTR comprises SEQ ID NO: 59. In some embodiments, the binary vector comprises a promoter containing a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 18. In some embodiments, the promoter is operatively linked to a 5' UTR containing the nucleic acid sequence of SEQ ID NO: 60. In some embodiments, the promoter comprises SEQ ID NO: 18. In some embodiments, the promoter comprises SEQ ID NO: 18, and the 5' UTR contains SEQ ID NO: 60. In some embodiments, the binary vector comprises a promoter containing a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 19. In some embodiments, the promoter is operatively linked to a 5' UTR containing the nucleic acid sequence of SEQ ID NO: 61. In some embodiments, the promoter comprises SEQ ID NO: 19. In some embodiments, the promoter comprises SEQ ID NO: 19, and the 5' UTR comprises SEQ ID NO: 61. In some embodiments, the binary vector comprises a promoter containing a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 1. In some embodiments, the promoter is operatively linked to a 5' UTR containing the nucleic acid sequence of SEQ ID NO: 43.In some embodiments, the promoter comprises SEQ ID NO: 1. In some embodiments, the promoter comprises SEQ ID NO: 1, and the 5' UTR comprises SEQ ID NO: 43. In some embodiments, the binary vector comprises a promoter comprising a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 20. In some embodiments, the promoter is operatively linked to a 5' UTR comprising a nucleic acid sequence comprising SEQ ID NO: 62. In some embodiments, the promoter comprises SEQ ID NO: 20. In some embodiments, the promoter comprises SEQ ID NO: 20, and the 5' UTR comprises SEQ ID NO: 62. In some embodiments, the binary vector comprises a promoter comprising a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 21. In some embodiments, the promoter is operatively linked to a 5' UTR containing the nucleic acid sequence of SEQ ID NO: 63. In some embodiments, the promoter contains SEQ ID NO: 21. In some embodiments, the promoter contains SEQ ID NO: 21, and the 5' UTR contains SEQ ID NO: 63. In some embodiments, the binary vector contains a promoter that contains a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 22. In some embodiments, the promoter is operatively linked to a 5' UTR containing the nucleic acid sequence of SEQ ID NO: 64. In some embodiments, the promoter contains SEQ ID NO: 22. In some embodiments, the promoter contains SEQ ID NO: 22, and the 5' UTR contains SEQ ID NO: 64. In some embodiments, the binary vector comprises a promoter containing a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 23. In some embodiments, the promoter is operatively linked to a 5' UTR containing a nucleic acid sequence of SEQ ID NO: 65. In some embodiments, the promoter contains SEQ ID NO: 23. In some embodiments, the promoter contains SEQ ID NO: 23, and the 5' UTR contains SEQ ID NO: 65. In some embodiments, the binary vector comprises a promoter containing a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 6.In some embodiments, the promoter is operatively linked to a 5' UTR containing the nucleic acid sequence of SEQ ID NO: 48. In some embodiments, the promoter contains SEQ ID NO: 6. In some embodiments, the promoter contains SEQ ID NO: 6, and the 5' UTR contains SEQ ID NO: 48. In some embodiments, the binary vector contains a promoter that contains a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 24. In some embodiments, the promoter is operatively linked to a 5' UTR containing the nucleic acid sequence of SEQ ID NO: 66. In some embodiments, the promoter contains SEQ ID NO: 24. In some embodiments, the promoter contains SEQ ID NO: 24, and the 5' UTR contains SEQ ID NO: 66.

[0010] In some embodiments, the reporter gene is selected from genes encoding fluorescent proteins, genes encoding bioluminescent proteins, and genes encoding pigment proteins. In some embodiments, the reporter gene encodes a fluorescent protein. In some embodiments, the reporter gene encodes bFLO_GFP. In some embodiments, the binary vector is used in soybean (soybean, Glycine max Gene regulatory activity is exhibited in plants.

[0011] In some embodiments, this disclosure provides plant cells comprising nucleic acid molecules, wherein the nucleic acid molecules comprise: a) a promoter comprising a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with a nucleic acid sequence selected from SEQ ID NO: 1-42; and b) a reporter gene, wherein the promoter is operatively linked to the reporter gene such that, in response to exposure to a stressor, the promoter induces expression of the reporter gene in the plant. In some embodiments, the nucleic acid molecule further comprises a 5' UTR comprising a nucleic acid sequence selected from SEQ ID NO: 43-79, wherein the nucleic acid sequence encoding the 5' UTR is operatively linked to a nucleic acid sequence encoding the promoter. In some embodiments, the reporter gene is selected from genes encoding fluorescent proteins, genes encoding bioluminescent proteins, and genes encoding pigment proteins. In some embodiments, the reporter gene encodes a fluorescent protein. In some embodiments, the reporter gene encodes bFLO_GFP. In some embodiments, the nucleic acid molecule is used in soybean (soybean, Glycine max Gene regulatory activity is exhibited in plants.

[0012] In some embodiments, the plant cell comprises a nucleic acid molecule, wherein the nucleic acid molecule comprises a promoter, said promoter comprising a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 4. In some embodiments, the promoter is operatively linked to a 5' UTR comprising a nucleic acid sequence comprising SEQ ID NO: 46. In some embodiments, the promoter comprises SEQ ID NO: 4. In some embodiments, the promoter comprises SEQ ID NO: 4, and the 5' UTR comprises SEQ ID NO: 46. In some embodiments, the plant cell comprises a nucleic acid molecule, wherein the nucleic acid molecule comprises a promoter, said promoter comprising a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 14. In some embodiments, the promoter is operatively linked to a 5' UTR comprising a nucleic acid sequence comprising SEQ ID NO: 56. In some embodiments, the promoter comprises SEQ ID NO: 14. In some embodiments, the promoter comprises SEQ ID NO: 14, and the 5' UTR comprises SEQ ID NO: 56. In some embodiments, the plant cell comprises a nucleic acid molecule containing a promoter that comprises a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 15. In some embodiments, the promoter is operatively linked to a 5' UTR containing a nucleic acid sequence of SEQ ID NO: 57. In some embodiments, the promoter comprises SEQ ID NO: 15. In some embodiments, the promoter comprises SEQ ID NO: 15, and the 5' UTR comprises SEQ ID NO: 57. In some embodiments, the plant cell comprises a nucleic acid molecule containing a promoter that comprises a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 16. In some embodiments, the promoter is operatively linked to a 5' UTR containing the nucleic acid sequence of SEQ ID NO: 58. In some embodiments, the promoter contains SEQ ID NO: 16. In some embodiments, the promoter contains SEQ ID NO: 16, and the 5' UTR contains SEQ ID NO: 58. In some embodiments, the plant cell contains a nucleic acid molecule containing a promoter that contains a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 17.In some embodiments, the promoter is operatively linked to a 5' UTR containing the nucleic acid sequence of SEQ ID NO: 59. In some embodiments, the promoter contains SEQ ID NO: 17. In some embodiments, the promoter contains SEQ ID NO: 17, and the 5' UTR contains SEQ ID NO: 59. In some embodiments, the plant cell contains a nucleic acid molecule containing a promoter that contains a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 18. In some embodiments, the promoter is operatively linked to a 5' UTR containing the nucleic acid sequence of SEQ ID NO: 60. In some embodiments, the promoter contains SEQ ID NO: 18. In some embodiments, the promoter contains SEQ ID NO: 18, and the 5' UTR contains SEQ ID NO: 60. In some embodiments, the plant cell comprises a nucleic acid molecule, wherein the nucleic acid molecule comprises a promoter, said promoter comprising a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 19. In some embodiments, the promoter is operatively linked to a 5' UTR comprising a nucleic acid sequence comprising SEQ ID NO: 61. In some embodiments, the promoter comprises SEQ ID NO: 19. In some embodiments, the promoter comprises SEQ ID NO: 19, and the 5' UTR comprises SEQ ID NO: 61. In some embodiments, the plant cell comprises a nucleic acid molecule, wherein the nucleic acid molecule comprises a promoter, said promoter comprising a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 1. In some embodiments, the promoter is operatively linked to a 5' UTR comprising a nucleic acid sequence comprising SEQ ID NO: 43. In some embodiments, the promoter comprises SEQ ID NO: 1. In some embodiments, the promoter comprises SEQ ID NO: 1, and the 5' UTR comprises SEQ ID NO: 43. In some embodiments, the plant cell comprises a nucleic acid molecule, wherein the nucleic acid molecule comprises a promoter, said promoter comprising a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 20. In some embodiments, the promoter is operatively linked to a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 62. In some embodiments, the promoter comprises SEQ ID NO: 20.In some embodiments, the promoter comprises SEQ ID NO: 20, and the 5' UTR comprises SEQ ID NO: 62. In some embodiments, the plant cell comprises a nucleic acid molecule, wherein the nucleic acid molecule comprises a promoter, said promoter comprising a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 21. In some embodiments, the promoter is operatively linked to a 5' UTR comprising a nucleic acid sequence comprising SEQ ID NO: 63. In some embodiments, the promoter comprises SEQ ID NO: 21. In some embodiments, the promoter comprises SEQ ID NO: 21, and the 5' UTR comprises SEQ ID NO: 63. In some embodiments, the plant cell comprises a nucleic acid molecule, wherein the nucleic acid molecule comprises a promoter, said promoter comprising a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 22. In some embodiments, the promoter is operatively linked to a 5' UTR containing the nucleic acid sequence of SEQ ID NO: 64. In some embodiments, the promoter contains SEQ ID NO: 22. In some embodiments, the promoter contains SEQ ID NO: 22, and the 5' UTR contains SEQ ID NO: 64. In some embodiments, the plant cell contains a nucleic acid molecule containing a promoter that contains a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 23. In some embodiments, the promoter is operatively linked to a 5' UTR containing the nucleic acid sequence of SEQ ID NO: 65. In some embodiments, the promoter contains SEQ ID NO: 23. In some embodiments, the promoter contains SEQ ID NO: 23, and the 5' UTR contains SEQ ID NO: 65. In some embodiments, the plant cell contains a nucleic acid molecule, wherein the nucleic acid molecule contains a promoter, said promoter containing a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 6. In some embodiments, the promoter is operatively linked to a 5' UTR containing a nucleic acid sequence of SEQ ID NO: 48. In some embodiments, the promoter contains SEQ ID NO: 6. In some embodiments, the promoter contains SEQ ID NO: 6, and the 5' UTR contains SEQ ID NO: 48.In some embodiments, the plant cell contains a nucleic acid molecule, wherein the nucleic acid molecule contains a promoter comprising a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 24. In some embodiments, the promoter is operatively linked to a 5' UTR comprising the nucleic acid sequence of SEQ ID NO: 66. In some embodiments, the promoter comprises SEQ ID NO: 24. In some embodiments, the promoter comprises SEQ ID NO: 24, and the 5' UTR comprises SEQ ID NO: 66. In some embodiments, the reporter gene is selected from genes encoding fluorescent proteins, genes encoding bioluminescent proteins, and genes encoding pigment proteins. In some embodiments, the reporter gene encodes a fluorescent protein. In some embodiments, the reporter gene encodes bFLO_GFP. In some embodiments, the nucleic acid molecule is in soybean (soybean, ...). Glycine max Gene regulatory activity is exhibited in plants.

[0013] In some embodiments, this disclosure provides a plant comprising nucleic acid molecules as described herein, binary vectors as described herein, and / or plant cells as described herein. In some embodiments, the plant is soybean (soybean, Glycine max )plant.

[0014] In some embodiments, this disclosure provides a grain comprising nucleic acid molecules as described herein, binary vectors as described herein, and / or plant cells as described herein. Brief description of the attached diagram Figures 1A-1N Representative images of plants transformed with promoter-reporter pairs, and of plants whose expression efficiency in response to fungal stress was tested at 0 h and 72 h after exposure to fungal stress, are depicted.

[0016] Figures 2A-2H Representative images of plants transformed with promoter-reporter pairs containing Gm chitinase and Gm chitinase 5' UTR were depicted, and their expression efficiency in response to fungal stress was tested at 0 h and 72 h after exposure to fungal stress.

[0017] Detailed description As used herein, "nucleic acid" refers to a polymeric compound comprising covalently linked nucleotides, said nucleotides containing native subunits (e.g., purine or pyrimidine bases). In some embodiments, the nucleic acid molecule is a polynucleotide molecule. In some embodiments, the nucleic acid molecule contains a genetic material. Purine bases include adenine and guanine, and pyrimidine bases include uracil, thymine, and cytosine. Nucleic acid molecules include ribonucleic acid (RNA) and deoxyribonucleic acid (DNA), which include cDNA, genomic DNA, and synthetic DNA, any one of which is single-stranded or double-stranded.

[0018] As used herein, the term “DNA” or “DNA molecule” refers to a double-stranded DNA molecule of genomic or synthetic origin, that is, a polymer or polynucleotide molecule of deoxyribonucleotide bases read from the 5' (upstream) end to the 3' (downstream) end. As used herein, the term “DNA sequence” refers to the nucleotide sequence of a DNA molecule.

[0019] As used herein, the term "sequence identity" refers to the degree to which two optimally aligned nucleic acid sequences are identical. Optimal sequence alignments are generated by manually aligning two sequences (e.g., a reference sequence and another sequence) to maximize the number of nucleotide matches in sequence alignments with appropriate internal nucleotide insertions, deletions, or vacancies.

[0020] As used herein, the terms “sequence identity percentage” or “identity percentage” or “%identity” are identity scores multiplied by 100. The “identity score” of a sequence that is best aligned to a reference sequence is the number of nucleotide matches in the best alignment divided by the total number of nucleotides in the reference sequence, for example, the total number of nucleotides in the full length of the reference sequence.

[0021] The term "transgenic" refers to a nucleic acid sequence that has been transferred into a cell (e.g., a plant cell). Transgenics contain nucleic acids and, in some embodiments, are incorporated into cells by any method disclosed herein or known to those skilled in the art, including but not limited to Agrobacterium-mediated transformation and transformation by particle bombardment (e.g., using a particle / gene gun).

[0022] As used herein, the term “expression cassette” refers to a different component or segment of vector DNA to be expressed by transformed cells, which consists of a gene and one or more regulatory elements.

[0023] As used herein, the term "operationally ligated" refers to the association of two or more nucleic acid molecules on a single nucleic acid fragment such that the function of one is affected by the function of the other.

[0024] As used herein, a “regulatory element” is a DNA molecule with gene regulatory activity, that is, a molecule with the ability to influence the transcription and / or translation of operably linked transcribed nucleic acid molecules. Therefore, the term “gene regulatory activity” refers to the ability to influence the expression pattern of a nucleic acid molecule by affecting its transcription and / or translation. Consequently, isolated regulatory elements (such as promoters and leader sequences) that function in plants can be used to modify plant phenotypes through genetic engineering methods.

[0025] As used herein, the term “promoter” generally refers to a DNA molecule that participates in the recognition and binding of RNA polymerase II and other proteins (trans-acting transcription factors) to initiate transcription.

[0026] As used herein, the term "5' UTR" refers to the 5' untranslated region (5'UTR) of a DNA molecule used to produce mRNA molecules during transcription. The 5' UTR can be operatively linked to and located upstream of a nucleic acid molecule, and can contain polynucleotides that provide regulatory signals, including but not limited to signals capable of influencing transcription and translation. The 5' UTR may also be referred to as a "leader sequence."

[0027] As used herein, the term “3' UTR” refers to the 3' untranslated region (3'UTR) of a DNA molecule used to produce mRNA molecules during transcription. The 3' UTR may be operatively linked to and located downstream of a nucleic acid molecule and may contain polynucleotides that provide polyadenylation signals and other regulatory signals, including but not limited to signals capable of influencing transcription, mRNA processing, or gene expression.

[0028] As used herein, the term "enhancer" or "enhancer element" refers to a cis-acting transcriptional regulatory element, i.e., a cis-element, which imparts one aspect of the overall expression pattern of an operably linked nucleic acid sequence, but is generally insufficient alone to drive transcription. Unlike promoters, enhancer elements typically do not include a transcription start site (TSS) or TATA box or equivalent sequence. Promoters may naturally contain one or more enhancer elements that influence the transcription of operably linked nucleic acid sequences. Isolated enhancer elements may also be fused to promoters. Promoters may contain one or more enhancer elements that influence the transcription of operably linked nucleic acid molecules. Enhancers may be located upstream or downstream of the operably linked nucleic acid molecule.

[0029] As used herein, the term "operably linked" refers to the connection of a first molecule to a second molecule, wherein the molecules are arranged such that the first molecule influences the function of the second molecule. The two molecules may or may not be part of a single, continuous molecule, and may or may not be adjacent. For example, in some embodiments, a promoter is operably linked to a nucleic acid molecule such that the promoter regulates the transcription of the nucleic acid molecule in the cell.

[0030] As used herein, the term "reporter gene" refers to a gene that encodes a reporter protein. The term "reporter protein" refers to a protein that produces a phenotypic change, phenomenon, or signal that can be identified on a visible spectrum. Reporter proteins can influence such phenotypic changes, phenomena, or signals through enzymatic activity or other mechanisms known to those skilled in the art.

[0031] As used herein, the term "pigment protein" refers to proteins that are pigment molecules, as well as proteins that are directly or indirectly involved in the synthesis of pigment molecules. Pigment proteins alter the pigment composition within plant cells.

[0032] As used herein, the term "grain" refers to a fruit or caryopsis harvested for human and / or animal consumption. A grain may contain an attached husk, or the husk may be detached.

[0033] As used herein, the term “agronomic trait” refers to a plant and / or crop characteristic that affects the yield, quality, and / or resistance to biotic and abiotic stresses of a plant and / or crop.

[0034] As used herein, the term "construct" means any recombinant nucleic acid molecule, such as plasmids, granules, viruses, autonomously replicating nucleic acid molecules, bacteriophages, or linear or circular single-stranded or double-stranded DNA or RNA polynucleotide molecules, derived from any source, capable of genome integration or autonomous replication, containing one or more nucleic acid molecules that have been functionally and operably linked (i.e., operably linked). As used herein, the term "vector" means any recombinant nucleic acid construct that can be used for transformation purposes (i.e., introducing heterologous DNA into host cells). This term includes expression cassettes isolated from any of the aforementioned molecules.

[0035] The term “transformation” refers to the introduction of nucleic acids into a recipient host. As used herein, the term “host” refers to bacteria, fungi, or plants, including any cell, tissue, organ, or progeny of bacteria, fungi, or plants.

[0036] All publications, patents, and patent applications cited in this specification are incorporated herein by reference in their entirety as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated by reference. Furthermore, each cited publication, patent, or patent application is incorporated herein by reference in its entirety to disclose and describe the subject matter relating to the cited publication.

[0037] Before further describing the present technology, it should be understood that the present technology is not limited to the specific embodiments described, and therefore, variations are certainly possible. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be restrictive, as the scope of this disclosure will be defined only by the appended claims. It should also be understood that the headings used herein are not restrictive and are intended only to guide the reader, but the subject matter generally applies to the technology disclosed herein.

[0038] promoter Transforming host cells with transgenes and / or constructs does not always result in robust expression of nucleic acid molecules, and specific promoters operatively linked to nucleic acid molecules (e.g., transgenes) can significantly influence subsequent transcription (expression) of nucleic acid molecules (e.g., transgenes) in host cells. This disclosure provides a first nucleic acid sequence encoding a promoter that, when operatively linked to a second nucleic acid molecule, induces increased expression of the second nucleic acid molecule in host cells. In some embodiments, the promoters described herein induce transcription (expression) of nucleic acid molecules in response to plant stressors. In some embodiments, plant stressors are abiotic stressors. Abiotic stressors include, but are not limited to, environmental factors such as water, temperature, wind, nutrient deficiency, and salinity. Abiotic stressors may also include chemical stressors associated with and caused by the application of various chemical treatments. In some embodiments, plant stressors are biotic stressors. Biological stressors include, but are not limited to, insects, bacteria, viruses, fungi, and other plants.

[0039] Table 1 provides exemplary nucleic acid sequences encoding promoters suitable for driving transgenic expression as described herein.

[0040] Table 1. Promoter sequences. In some embodiments, the nucleic acid molecule encoding the promoter described herein comprises a promoter selected from SEQ ID NO: 1-42. In some embodiments, the nucleic acid molecule encoding the promoter described herein comprises a promoter having the nucleic acid sequence of SEQ ID NO: 4. In some embodiments, the nucleic acid molecule encoding the promoter described herein comprises a promoter having the nucleic acid sequence of SEQ ID NO: 14. In some embodiments, the nucleic acid molecule encoding the promoter described herein comprises a promoter having the nucleic acid sequence of SEQ ID NO: 15. In some embodiments, the nucleic acid molecule encoding the promoter described herein comprises a promoter having the nucleic acid sequence of SEQ ID NO: 16. In some embodiments, the nucleic acid molecule encoding the promoter described herein comprises a promoter having the nucleic acid sequence of SEQ ID NO: 17. In some embodiments, the nucleic acid molecule encoding the promoter described herein comprises a promoter having the nucleic acid sequence of SEQ ID NO: 18. In some embodiments, the nucleic acid molecule encoding the promoter described herein comprises a promoter having the nucleic acid sequence of SEQ ID NO: 19. In some embodiments, the nucleic acid molecule encoding the promoter described herein comprises a promoter having the nucleic acid sequence of SEQ ID NO: 1. In some embodiments, the nucleic acid molecule encoding the promoter described herein comprises a promoter having the nucleic acid sequence of SEQ ID NO: 20. In some embodiments, the nucleic acid molecule encoding the promoter described herein comprises a promoter having the nucleic acid sequence of SEQ ID NO: 21. In some embodiments, the nucleic acid molecule encoding the promoter described herein comprises a promoter having the nucleic acid sequence of SEQ ID NO: 22. In some embodiments, the nucleic acid molecule encoding the promoter described herein comprises a promoter having the nucleic acid sequence of SEQ ID NO: 23. In some embodiments, the nucleic acid molecule encoding the promoter described herein comprises a promoter having the nucleic acid sequence of SEQ ID NO: 6. In some embodiments, the nucleic acid molecule encoding the promoter described herein comprises a promoter having the nucleic acid sequence of SEQ ID NO: 24.

[0041] In some implementations, the nucleic acid molecule encoding the promoter as described herein has at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity with a sequence selected from SEQ ID NO: 1-42.

[0042] The promoters described herein can be operatively linked to one or more other regulatory elements described herein or known in the art. For example, in some embodiments, the promoters described herein can be operatively linked to an enhancer or a UTR (5' and / or 3'). In some embodiments, the promoters described herein are operatively linked to a 5' UTR. Exemplary nucleic acid sequences encoding 5' UTRs are provided in Table 2 below. In some embodiments, the promoters described herein are operatively linked to a 5' UTR having a nucleic acid sequence selected from one of SEQ ID NO: 43-79. Furthermore, in some embodiments, the promoters described herein can be operatively linked to a transforming enhancer sequence (TBS) or other elements that enhance promoter activity.

[0043] Table 2. 5' UTR sequence. In some embodiments, the nucleic acid molecule encoding the promoter described herein comprises the nucleic acid sequence SEQ ID NO: 4 (GmPR4), which is operatively linked to the nucleic acid sequence SEQ ID NO: 46 (GmPR4 5' UTR). In some embodiments, the nucleic acid molecule encoding the promoter described herein comprises the nucleic acid sequence SEQ ID NO: 14 (SolCHI4), which is operatively linked to the nucleic acid sequence SEQ ID NO: 56 (SolCHI4 5' UTR). In some embodiments, the nucleic acid molecule encoding the promoter described herein comprises the nucleic acid sequence SEQ ID NO: 15 (GmPR2), which is operatively linked to the nucleic acid sequence SEQ ID NO: 57 (GmPR2 5' UTR). In some embodiments, the nucleic acid molecule encoding the promoter described herein comprises the nucleic acid sequence SEQ ID NO: 16 (GmMFP), which is operatively linked to the nucleic acid sequence SEQ ID NO: 58 (GmMFP 5' UTR). In some embodiments, the nucleic acid molecule encoding the promoter described herein comprises the nucleic acid sequence of SEQ ID NO: 17 (Gm chitinase), which is operatively linked to the nucleic acid sequence of SEQ ID NO: 59 (Gm chitinase 5' UTR). In some embodiments, the nucleic acid molecule encoding the promoter described herein comprises the nucleic acid sequence of SEQ ID NO: 18 (GmCHS7), which is operatively linked to the nucleic acid sequence of SEQ ID NO: 60 (GmCHS7 5' UTR). In some embodiments, the nucleic acid molecule encoding the promoter described herein comprises the nucleic acid sequence of SEQ ID NO: 19 (GmFDH), which is operatively linked to the nucleic acid sequence of SEQ ID NO: 61 (GmFDH 5' UTR). In some embodiments, the nucleic acid molecule encoding the promoter described herein comprises the nucleic acid sequence of SEQ ID NO: 1 (GmCHIT2), which is operatively linked to the nucleic acid sequence of SEQ ID NO: 43 (GmCHIT2 5' UTR). In some implementations, the nucleic acid molecule encoding the promoter as described herein comprises the nucleic acid sequence SEQ ID NO: 20 (GmPI4), which is operatively linked to the nucleic acid sequence SEQ ID NO: 62 (GmPI4 5' UTR).In some embodiments, the nucleic acid molecule encoding the promoter described herein comprises the nucleic acid sequence SEQ ID NO: 21 (GmPR3V2), which is operatively linked to the nucleic acid sequence SEQ ID NO: 63 (GmPR3V2 5' UTR). In some embodiments, the nucleic acid molecule encoding the promoter described herein comprises the nucleic acid sequence SEQ ID NO: 22 (GmPI39), which is operatively linked to the nucleic acid sequence SEQ ID NO: 64 (GmPI39 5' UTR). In some embodiments, the nucleic acid molecule encoding the promoter described herein comprises the nucleic acid sequence SEQ ID NO: 23 (GmPR1), which is operatively linked to the nucleic acid sequence SEQ ID NO: 65 (GmPR1 5' UTR). In some embodiments, the nucleic acid molecule encoding the promoter described herein comprises the nucleic acid sequence SEQ ID NO: 6 (GmPI09), which is operatively linked to the nucleic acid sequence SEQ ID NO: 48 (GmPI09 5' UTR). In some implementations, the nucleic acid molecule encoding the promoter as described herein comprises the nucleic acid sequence SEQ ID NO: 24 (GmPPO12), which is operatively linked to the nucleic acid sequence SEQ ID NO: 66 (GmPPO12 5' UTR).

[0044] Report As described herein, this disclosure provides a first nucleic acid molecule encoding a promoter, operatively linked to a second nucleic acid molecule. In some embodiments, the second nucleic acid molecule encodes a gene of interest. In some embodiments, the gene of interest is a gene that provides a useful agronomic trait. In some embodiments, the gene of interest that provides a useful agronomic trait is a reporter. A reporter is a gene that is not normally present in the cell prior to transformation and includes a gene encoding a protein that induces intracellular phenotypic changes in response to transformation. The reporter gene may also include, for example, a gene encoding a protein that provides enzymatic activity within the cell. The reporter gene produces a signal detectable by a cellular observer.

[0045] In some embodiments, the signal generated by the reporter gene is detectable at a macroscopic scale. In some embodiments, the signal generated by the reporter is detectable at a microscopic scale. Suitable reporter genes are generally known to those skilled in the art and may include, but are not limited to, genes encoding fluorescent proteins, bioluminescent proteins, and pigment proteins that alter the pigment composition of plant cells, making phenotypic differences in the cells identifiable. Fluorescent proteins compatible with the constructs described herein include, but are not limited to, green fluorescent protein (GFP), yellow fluorescent protein (YFP), sky blue fluorescent protein (CFP), red fluorescent protein (RFP), orange fluorescent protein (OFP), and variants thereof (e.g., tdTomato, mCherry, DsRed, and bFLO-GFP1). Bioluminescent proteins compatible with the constructs described herein include, but are not limited to, luminescent proteins and luciferases and variants thereof. Luminescent proteins and luciferases include, but are not limited to, aequorin, berovin, firefly luciferase, oplophorus luciferase, gaussia luciferase, dinoflagellate luciferase, renin luciferase, and bacterial luciferase and their variants. Pigment proteins compatible with the constructs described herein include, but are not limited to, anthocyanins, betaine, carotenoids, and their variants.

[0046] In addition to the reporting materials described herein, it should be understood that the embodiments described herein may include any reporting materials known in the art.

[0047] This disclosure also provides additional regulatory elements operatively linked to a gene of interest (e.g., a reporter gene) and / or a promoter. These additional regulatory elements include those described herein, including but not limited to enhancers, 5' UTRs, and 3' UTRs.

[0048] binary construct In some embodiments, the constructs of the present invention can be provided as binary vector constructs comprising a first nucleic acid molecule encoding a promoter, the first nucleic acid molecule being operatively linked to a second nucleic acid molecule as described herein. In some embodiments, the binary construct comprises components derived from Agrobacterium tumefaciens (… Agrobacterium tumefaciensThe tumor-inducing plasmid (Ti plasmid) isolated from the organism comprises the right boundary (RB T-DNA repeat sequence) and left boundary (LB T-DNA repeat sequence) regions of the T-DNA, which, together with transfer molecules provided by Agrobacterium tumefaciens cells, allows the integration of the T-DNA into the genome of the plant cell (see, for example, U.S. Patent No. 6,603,061). The construct may also include plasmid backbone DNA segments commonly known in the art that provide replication function and antibiotic selection in bacterial cells, for example, including Escherichia coli (…). Escherichia coli The coding regions of replication origins, wide host-range replication origins (such as oriV or oriRi), replication initiators (e.g., RepA), stability proteins (e.g., StaA), and selection marker genes.

[0049] This disclosure also provides constructs that include additional regulatory elements, including but not limited to promoters, leader sequences, UTRs, introns, and transcription termination regions.

[0050] The constructs and vectors described herein may also include a transport peptide encoding a linker peptide that can be used to transport protein products to specific organelles, particularly to chloroplasts, leucoplasts, or other plastid organelles; mitochondria; peroxisomes; vacuoles; or extracellular locations. Uses of chloroplast transport peptides are described, for example, in U.S. Patents 5,188,642 and 5,728,925. Many chloroplast-localized proteins are expressed as precursors from nuclear genes and transported to chloroplasts via chloroplast transport peptides (CTPs). CTPs and other transport peptides are generally known in the art and can be used in the constructs described herein to improve the transport of expressed transgenes to specific intracellular locations.

[0051] Methods for preparing and using constructs, as well as host cells, can be applied in, for example, Molecular Cloning. A Laboratory Manual The third edition, Volumes 1, 2, and 3 (2000), JF Sambrook, DW Russell, and N. Irwin, Cold Spring Harbor Laboratory Press, can be found. Methods for preparing recombinant vectors particularly suitable for plant transformation may include, but are not limited to, those described in U.S. Patent Nos. 4,971,908; 4,940,835; 4,769,061; and 4,757,011. These types of vectors have also been reviewed in the scientific literature (see, for example, Rodriguez, et al.). Vectors: A Survey of Molecular Cloning Vectors and Their Uses,Butterworths, Boston, (1988) and Glick, et al. Methods in Plant Molecular Biology and Biotechnology CRC Press, Boca Raton, Fla. (1993)). Typical vectors for expressing nucleic acids in plants are well known in the art, and include, but are not limited to, vectors derived from Ti plasmids of Agrobacterium tumefaciens (Rogers, et al.). Methods in Enzymology 153: 253-277 (1987)).

[0052] Conversion and use of constructs This disclosure also relates to methods for producing transformed cells and plants comprising promoters operatively linked to nucleic acid molecules (e.g., transgenes) and / or constructs described herein.

[0053] In some implementations, the transformed cells are soybeans ( Glycine max ) cells. In some implementations, the plant being transformed is soybean ( Glycine max )plant.

[0054] Methods for introducing nucleic acid molecules into host cells typically include the following steps: selecting suitable host cells, transforming the host cells with a recombinant vector, and obtaining transformed host cells. Suitable methods include those described herein, such as bacterial infection (e.g., Agrobacterium), generation of binary bacterial artificial chromosome vectors, direct delivery of DNA (e.g., transformation via PEG, drying / inhibition-mediated DNA uptake, electroporation, stirring with silicon carbide fibers, and acceleration of DNA-coated particles, etc., as reviewed in Potrykus, et al., Ann. Rev. Plant Physiol. Plant Mol. Biol. 42: 205 (1991)). Host cells can be transformed with the transgenic and / or constructs described herein using any acceptable method for plant transformation.

[0055] The presence of the transgenes and / or constructs described herein can be analyzed in transformed plants, and expression levels and / or profiles can be assigned. Those skilled in the art will recognize many methods that can be used to analyze transformed plants. For example, methods for plant analysis include, but are not limited to, DNA blotting or RNA blotting, PCR-based methods, biochemical analysis, phenotypic screening methods, field evaluation, and immunodiagnostic assays.

[0056] Seeds of plants transformed with the transgenic and / or constructs described herein can be harvested from fertile transgenic plants and used to breed offspring generations of the transformed plants, including hybrid plant lines containing the transgenic and / or constructs described herein and expressing genes of agronomical interest.

[0057] Transgenic plants can pass on transgenes and / or constructs to their offspring. Offspring include any renewable plant parts or seeds containing transgenes and / or constructs derived from an ancestral plant. The transgenic plant is preferably homozygous for the transgene and / or construct and passes the nucleic acid molecule to all offspring through sexual reproduction. Offspring can grow from seeds produced by the transgenic plant. These additional plants can then be self-pollinated to produce true breeding lines of the plant. Among other things, gene expression in the offspring from these plants is assessed. Gene expression can be detected using several commonly used methods, such as Western blotting, RNA blotting, immunoprecipitation, and ELISA.

[0058] The following embodiments are provided by way of illustration and are not intended to limit the scope of this disclosure. Those skilled in the art will understand from this disclosure that many changes can be made to the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of this disclosure. Therefore, all content set forth or illustrated in the embodiments and drawings should be interpreted as illustrative rather than restrictive.

[0059] Example Example 1: Identification of genes of interest.

[0060] Analyze publicly available RNA-seq datasets to identify pathogen stress in soybeans ( Glycine max Genes induced in soybean plants were identified. The identified genes were sequenced based on fold changes in gene expression induction levels in response to pathogen stress. In addition to genes induced in soybean, genes induced in potato were also identified. Solanum tuberosum The genes identified in the analysis were included in this preliminary analysis. The genes identified in the analysis are provided in Table 3. Genes exhibiting the highest fold change in response to pathogen stress are located at the top of the table, and genes exhibiting the lowest fold change in response to pathogen stress are located at the bottom of the table.

[0061] Table 3. Genes of interest. Fourteen genes from Table 3 were selected, cloned, and transformed into soybean. Glycine max In plants, it is used for further testing in disease assays.

[0062] Example 2: Promoter testing in plants Testing of the promoters identified in Example 1 on soybean ( Glycine maxThe study was conducted in plants. The identified promoter was operatively ligated to the reporter gene and incorporated into the Ti plasmid described herein, and plants were transformed using various constructs for Agrobacterium-mediated transformation as described herein. Soybean plants were grown in 9×4 trays (36 wells) filled with peat-based soil and irrigated with a fertilizer mixture (Jack's 20-20-20) with an electrical conductivity (EC) of 0.4. Plants were also grown in a growth chamber under conditions using 400 µmol / m³ of fertilizer. 2 LED lights were used for a 16-hour / 8-hour light-dark cycle, with a temperature of 24°C during the daytime (light) period and 22°C during the nighttime (dark) period. While plants with at least one trifoliate leaflet containing a leaflet two inches long and one inch wide were selected for further testing, plants exhibiting stress or disease (e.g., yellowing or small leaves) were excluded from further testing. Six-well test plates were prepared by inserting pre-cut pieces of floral foam / rock wool into the holes and wetting the foam / rock wool with sufficient tap water to create a pool below / around the foam / rock wool (but not submerging it).

[0063] Alternaria ( Alternaria alternata A fungal stressor was prepared by culturing the fungus on a PDA plate at 27°C in ambient light for up to two weeks before inoculation preparation to fresh potato dextrose agar (PDA) plates or potato dextrose broth (PDB). For liquid fungal growth, 6 g of powdered PDB was mixed in 250 mL of distilled water, and the reconstituted broth was added to a 500 mL wide-mouth culture bottle. A piece of coarse cotton cloth was folded three times and wedged into the bottle neck to form a tight seal. A piece of aluminum foil was folded three times and shaped above the bottle neck to make a cap for the bottle. The bottle was then autoclaved at 121°C for 20–35 minutes with the drain valve closed. The autoclaved medium was then inoculated with three to four pieces of Alternaria alternata grown on ½ cm square plates by aseptically transferring cut fungal slices to the liquid medium bottle. The inoculated medium bottle was then placed in a shaker at 28°C for five days.

[0064] To prepare the fungal solution for treatment application, mycelium was filtered from the liquid culture through four layers of coarse cotton cloth using a Buchner funnel and a vacuum pump. The filtered mycelium was rinsed at least five times with sterile distilled water, and after rinsing, the mycelium was squeezed with gloved hands to remove and dehydrate it. The mycelium was then weighed and stirred in a mixer (using a fruit puree setting) with sufficient sterile distilled water for two minutes to achieve a final concentration of 1% m / v mycelium. Tween was added to the mycelium solution in an amount sufficient to produce a 0.1% Tween:fungal inoculum solution (i.e., Tween volume (µL) = mycelium solution volume (mL) × 0.001). The fungal inoculum solution was stored at 4°C until needed.

[0065] Ethephon treatment for plants is prepared by adding 300 µL of Florel brand plant growth regulator (3.9% ethephon w / v) to a spray bottle containing 500 mL of DI water, providing a final solution of 0.002% ethephon. Add 500 µL of Tween-20 to this solution to bring the final Tween-20 concentration to 0.1%. Store the solution at room temperature (approximately 22°C) until use. Simulated ethephon treatment is prepared by mixing 500 mL of DI water with 500 µL of Tween-20 (final concentration 0.1%), and this solution is also stored at room temperature until use.

[0066] To perform this assay, two cuts were made at the median leaflet of the trifoliate leaves of the selected plants using clean scissors. The cut leaves were placed adaxially upward in a 6-well tray with pre-moistened floral foam / rock wool. Each well was treated by spraying and thoroughly wetting the exposed leaf surface to perform the specified treatment, and then a cover was placed on each plate after treatment. Initial images (time point 0) were taken, and the plates were placed in a germination chamber set to a constant 23°C and a 16-hour / 8-hour light-dark cycle. The plants were subsequently imaged 72 hours after treatment. Images were captured on a custom-made fluorescence imager. Excitation light for green fluorescent protein was generated by four Luxeon Z blue LEDs in tandem and filtered using a 472 / 36 bandpass filter. Images were acquired using a Basler acA3088-57uc camera equipped with an MVL8M23 – 8 mm EFL, f / 1.4 lens for a 2 / 3” C mount, and a built-in 520 / 36 bandpass filter. Quantitative fluorescence measurements were determined using ImageJ.

[0067] Table 4 provides the reporter gene induction using the promoters identified in Example 1 after exposure to fungal stress as described above. Average final brightness, average initial induction, final brightness count, maximum final brightness, and maximum initial induction are reported in Table 4. Initial induction was measured by dividing the brightness recorded in the 72-hour image by the brightness recorded in the 0-hour image. Brightness was measured as counts per second (0–255) and was a measure of the intensity of each of the three color channels (red, green, and blue) for each pixel in the image. To calculate leaf brightness, a boundary was drawn around the leaf. The brightest pixels within the boundary, comprising 10% of the total pixels within the boundary, were identified, and the average brightness of these pixels was calculated. This average was calculated only on the color channel most closely corresponding to the reporter color (e.g., the green channel for bFLO GFP and the red channel for TdTomato RFP). Initial fold change was calculated by dividing the brightness at the final measurement time point (typically 72 hours post-inoculation) by the brightness at the initial time point (typically 0 hours post-inoculation).

[0068] Table 4. The efficacy of different promoters in response to fungal stressors on reporter expression. Surprisingly, several promoters that exhibited low fold changes in gene expression induction levels in response to plant stressors in Example 1 showed the highest mean brightness measurements when the efficacy of constructs containing these promoters was determined in plants (see, for example, GmPPO, GmPR2, GmPR4, and GmCYP93C promoters), indicating that these promoters robustly induced the expression of operatively linked reporter genes in response to fungal stressors in soybean. Figures 1A-1N Representative images of the plant at time points 0 and 72 hours are shown. Figure 1A and Figure 1B The reporter gene induction at 0 h and 72 h is shown when the reporter gene is operatively linked to the GmCHIT2 promoter. Figure 1B Clearly, 72 hours after treatment, the GmCHIT2 promoter drove reporter gene expression at a level higher in plants than in plants containing other test promoters operatively linked to the same reporter gene. Figure 1C and Figure 1D The reporter gene induction at 0 h and 72 h is shown when the reporter gene is operatively linked to the GmPR4 promoter. As these figures illustrate, plants with the GmPR4 promoter exhibit robust expression of the reporter gene 72 h after treatment with the fungal stressor. Figure 1E and Figure 1F The reporter gene induction at 0 h and 72 h is shown when the reporter gene is operatively ligated to the GmCYP93C promoter. Figure 1G and Figure 1H The reporter gene induction at 0 h and 72 h is shown when the reporter gene is operatively linked to the GmPPO promoter. Notably, plants containing the GmPPO promoter operatively linked to the reporter gene exhibit robust expression of the reporter gene almost immediately after treatment with the fungal stressor, and this expression persists until 72 h post-treatment. Figure 1I and Figure 1J The reporter gene induction at 0 h and 72 h is shown when the reporter gene is operatively linked to the SolCHI4 promoter. At 72 h following treatment with the fungal stressor, the SolCHI4 promoter drove robust expression of the reporter gene. Figure 1K and Figure 1LThe reporter gene induction at 0 h and 72 h is shown when the reporter gene is operatively linked to the GmPR2 promoter. The GmPR2 promoter rapidly drives reporter gene expression, exhibiting significant expression at the first time point, shortly after treatment with the fungal stressor. Furthermore, the intensity of reporter gene expression driven by the GmPR2 promoter increases at the 72 h time point. Figure 1M and Figure 1N The reporter gene induction at 0 hours and 72 hours is shown when the reporter gene is operatively linked to the GmPI09 promoter.

[0069] Unbound by any particular theory, the promoters described herein act as effective drivers of reporter gene expression in soybean, as evidenced by increased brightness and induction levels (see, for example, GmPR2, GmPR2, SolCHI4). Therefore, these promoters can function as important regulators of reporter gene expression in response to plant stressors, such as fungal stressors, and may be useful in the binary vectors described herein for plant transformation.

[0070] Example 3: In vivo promoter testing in soil-grown plants For soybeans grown in soil ( Glycine max Various promoters in the plant were tested. T0 plants (also referred to herein as “events”) were cultured to maturity after transformation with plasmids carrying any of the various promoters described herein. Six seeds were collected from each event and sown in two separate pots. The plants were then grown for 3 weeks to the V1 / V2 stage, one pot of which was treated with *Cercospora soybeani* (…). Cercospora sojina One pot was treated with fungal spores, and another pot was treated simulantly according to the method described herein. Reporter gene fluorescence in the pots was imaged at treatment time (0h) and 3 days after treatment (72h). Fluorescence was quantified from each image. Counts per second (CPS) represent the pixel intensity value in the image (also referred to herein as “counts”), scaled by the exposure time (in seconds) used for image acquisition. Images were collected as 8-bit images with pixel intensities ranging from 0 to 255 (inclusive). CPS quantification was determined by dividing the pixel intensity value (counts) by the exposure time (in seconds). Images were acquired in linear mode without post-acquisition scaling. An event was counted as a putative hit if the quantification met all three of the following criteria: (i) fluorescence value > CPS measured 3 days after treatment; (ii) CPS 3 days after fungal treatment greater than 1.5 times that at fungal treatment; and (iii) CPS 3 days after fungal treatment greater than 1.5 times that of the simulated treated plant at 72h after the simulated treatment.

[0071] Prior to treatment, plants were grown in a growth chamber to V1 / V2 under long-day conditions (16 hours light / 8 hours darkness, 24°C). Plants undergoing the simulated treatment were treated with water and 0.05% Tween-20 surfactant. Plants undergoing the fungal treatment were treated with water, surfactant, and *Cercospora soybeanis* spores at a concentration of ~150,000 spores / mL.

[0072] Table 5. The efficacy of different promoters in driving reporter gene fluorescence in response to fungal stressors in soil-grown plants. The soil-grown plant data generated and detailed in Table 5 reveal that several promoters identified in both Examples 1 and 2, as well as newly identified in this example, are particularly effective in meeting the putative hit criteria described herein. Notably, the GmPR4, GmCHS7, and Gm chitinase promoters are the most effective in meeting the putative hit criteria as described herein. Consistent with the putative hit data in Table 5, and as... Figures 2A-2H Clearly, plants subjected to simulated treatments containing the Gm chitinase promoter (SEQ ID NO: 17) and Gm chitinase 5' UTR (SEQ ID NO: 59) showed significant improvement at 0 h ( Figure 2B ) and 72h ( Figure 2D Compared to other plants, those containing the Gm chitinase promoter (SEQ ID NO: 17) and Gm chitinase 5' UTR (SEQ ID NO: 59) showed better results when treated with fungi at 0 h ( Figure 2F ) and 72h ( Figure 2H Increased reporter fluorescence was observed between the two groups. Therefore, the Gm chitinase promoter acts as an effective driver of reporter gene expression and can be used to effectively report the presence of fungi in whole plants grown in soil.

[0073] Unbound by any particular theory, as indicated by these soil-grown plant data, the promoters described herein act as efficient drivers of reporter gene expression in soybean, as evidenced by their ability to meet presumed hit criteria. Therefore, these data provide further evidence that these promoters can thus function as important regulators of reporter gene expression in response to plant stressors, such as fungal stressors, and can be useful in the binary vectors described herein for plant transformation, including, but not limited to, their ability to function significantly as reporters in whole soil-grown plants.

Claims

1. A nucleic acid molecule comprising: a. A promoter comprising a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with a nucleic acid sequence selected from SEQ ID NO: 1-42; and b. Reporter genes, The promoter is operatively linked to the reporter gene such that, in response to exposure to a stressor, the promoter induces the expression of the reporter gene in the plant.

2. The nucleic acid molecule according to claim 1, further comprising a 5' UTR, the 5' UTR comprising a nucleic acid sequence selected from SEQ ID NO:43-79, wherein the nucleic acid sequence encoding the 5' UTR is operatively linked to a nucleic acid sequence encoding the promoter.

3. The nucleic acid molecule according to claim 1, wherein the reporter gene is selected from genes encoding fluorescent proteins, genes encoding bioluminescent proteins, and genes encoding pigment proteins.

4. The nucleic acid molecule according to claim 3, wherein the reporter gene encodes a fluorescent protein.

5. The nucleic acid molecule according to claim 4, wherein the reporter gene encodes bFLO_GFP.

6. The nucleic acid molecule according to claim 1, wherein the nucleic acid molecule is in soybean (soybean, Glycine max Gene regulatory activity is exhibited in plants.

7. The nucleic acid molecule according to claim 1, wherein the promoter comprises a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:

4.

8. The nucleic acid molecule of claim 7, wherein the promoter is operatively linked to a 5' UTR comprising the nucleic acid sequence of SEQ ID NO:

46.

9. The nucleic acid molecule according to claim 7, wherein the promoter comprises SEQ ID NO:

4.

10. The nucleic acid molecule of claim 7, wherein the promoter comprises SEQ ID NO: 4 and is operatively linked to a 5' UTR comprising SEQ ID NO:

46.

11. The nucleic acid molecule according to claim 7, wherein the reporter gene is selected from genes encoding fluorescent proteins, genes encoding bioluminescent proteins, and genes encoding pigment proteins.

12. The nucleic acid molecule of claim 11, wherein the reporter gene encodes a fluorescent protein.

13. The nucleic acid molecule of claim 12, wherein the reporter gene encodes bFLO_GFP.

14. The nucleic acid molecule according to claim 7, wherein the nucleic acid molecule is in soybean (soybean, Glycine max Gene regulatory activity is exhibited in plants.

15. The nucleic acid molecule of claim 1, wherein the promoter comprises a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO:

17.

16. The nucleic acid molecule of claim 15, wherein the promoter is operatively linked to a 5' UTR comprising the nucleic acid sequence of SEQ ID NO:

59.

17. The nucleic acid molecule of claim 15, wherein the promoter comprises SEQ ID NO:

17.

18. The nucleic acid molecule of claim 15, wherein the promoter comprises SEQ ID NO: 17 and is operatively linked to a 5' UTR comprising SEQ ID NO:

59.

19. The nucleic acid molecule of claim 15, wherein the reporter gene is selected from genes encoding fluorescent proteins, genes encoding bioluminescent proteins, and genes encoding pigment proteins.

20. The nucleic acid molecule of claim 19, wherein the reporter gene encodes a fluorescent protein.

21. The nucleic acid molecule of claim 20, wherein the reporter gene encodes bFLO_GFP.

22. The nucleic acid molecule according to claim 15, wherein the nucleic acid molecule is in soybean (soybean, Glycine max Gene regulatory activity is exhibited in plants.

23. The nucleic acid molecule according to claim 1, wherein the promoter comprises a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO:

18.

24. The nucleic acid molecule of claim 23, wherein the promoter is operatively linked to a 5' UTR comprising the nucleic acid sequence of SEQ ID NO:

60.

25. The nucleic acid molecule of claim 23, wherein the promoter comprises SEQ ID NO:

18.

26. The nucleic acid molecule of claim 23, wherein the promoter comprises SEQ ID NO: 18 and is operatively linked to a 5' UTR comprising SEQ ID NO:

60.

27. The nucleic acid molecule of claim 23, wherein the reporter gene is selected from genes encoding fluorescent proteins, genes encoding bioluminescent proteins, and genes encoding pigment proteins.

28. The nucleic acid molecule of claim 27, wherein the reporter gene encodes a fluorescent protein.

29. The nucleic acid molecule of claim 28, wherein the reporter gene encodes bFLO_GFP.

30. The nucleic acid molecule according to claim 23, wherein the nucleic acid molecule is in soybean (soybean, Glycine max Gene regulatory activity is exhibited in plants.

31. A binary vector for plant transformation, comprising: The nucleic acid sequence encodes: i) a promoter comprising a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with a nucleic acid sequence selected from SEQ ID NO: 1-42; and ii) a reporter gene, wherein the promoter is operatively linked to the reporter gene and induces expression of the reporter gene in a plant in response to exposure to a stressor.

32. The binary vector of claim 31, further comprising a 5' UTR, the 5' UTR comprising a nucleic acid sequence selected from SEQ ID NO:43-79, wherein the nucleic acid sequence encoding the 5' UTR is operatively linked to a nucleic acid sequence encoding the promoter.

33. The binary vector according to claim 31, wherein the reporter gene is selected from genes encoding fluorescent proteins, genes encoding bioluminescent proteins, and genes encoding pigment proteins.

34. The binary vector of claim 33, wherein the reporter gene encodes a fluorescent protein.

35. The binary vector according to claim 34, wherein the reporter gene encodes bFLO_GFP.

36. The binary carrier according to claim 31, wherein the nucleic acid molecule is in soybean (soybean, Glycine max Gene regulatory activity is exhibited in plants.

37. The binary vector according to claim 31, wherein the promoter comprises a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO:

4.

38. The binary vector of claim 37, wherein the promoter is operatively linked to a 5' UTR comprising the nucleic acid sequence of SEQ ID NO:

46.

39. The binary vector according to claim 37, wherein the promoter comprises SEQ ID NO:

4.

40. The binary carrier of claim 37, wherein the promoter comprises SEQ ID NO: 4 and is operatively connected to a 5' UTR comprising SEQ ID NO:

46.

41. The binary vector according to claim 37, wherein the reporter gene is selected from genes encoding fluorescent proteins, genes encoding bioluminescent proteins, and genes encoding pigment proteins.

42. The binary vector according to claim 41, wherein the reporter gene encodes a fluorescent protein.

43. The binary vector according to claim 42, wherein the reporter gene encodes bFLO_GFP.

44. The binary carrier according to claim 37, wherein the nucleic acid molecule is in soybean (soybean, Glycine max Gene regulatory activity is exhibited in plants.

45. The binary vector of claim 31, wherein the promoter comprises a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO:

17.

46. ​​The binary vector of claim 45, wherein the promoter is operatively linked to a 5' UTR comprising the nucleic acid sequence of SEQ ID NO:

59.

47. The binary vector of claim 45, wherein the promoter comprises SEQ ID NO:

17.

48. The binary carrier of claim 45, wherein the promoter comprises SEQ ID NO: 17 and is operatively connected to a 5' UTR comprising SEQ ID NO:

59.

49. The binary vector according to claim 45, wherein the reporter gene is selected from genes encoding fluorescent proteins, genes encoding bioluminescent proteins, and genes encoding pigment proteins.

50. The binary vector of claim 49, wherein the reporter gene encodes a fluorescent protein.

51. The binary vector according to claim 50, wherein the reporter gene encodes bFLO_GFP.

52. The binary carrier according to claim 45, wherein the nucleic acid molecule is in soybean (soybean, Glycine max Gene regulatory activity is exhibited in plants.

53. The binary vector according to claim 31, wherein the promoter comprises a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO:

18.

54. The binary vector of claim 53, wherein the promoter is operatively linked to a 5' UTR comprising the nucleic acid sequence of SEQ ID NO:

60.

55. The binary vector of claim 53, wherein the promoter comprises SEQ ID NO:

18.

56. The binary carrier of claim 53, wherein the promoter comprises SEQ ID NO: 18 and is operatively connected to a 5' UTR comprising SEQ ID NO:

60.

57. The binary vector according to claim 53, wherein the reporter gene is selected from genes encoding fluorescent proteins, genes encoding bioluminescent proteins, and genes encoding pigment proteins.

58. The binary vector of claim 57, wherein the reporter gene encodes a fluorescent protein.

59. The binary vector according to claim 58, wherein the reporter gene encodes bFLO_GFP.

60. The binary carrier according to claim 53, wherein the nucleic acid molecule is in soybean (soybean, Glycine max Gene regulatory activity is exhibited in plants.

61. A plant cell comprising nucleic acid molecules, said nucleic acid molecules comprising: a. A promoter comprising a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with a nucleic acid sequence selected from SEQ ID NO: 1-42; and b. Reporter genes, The promoter is operatively linked to the reporter gene such that, in response to exposure to a stressor, the promoter induces the expression of the reporter gene in the plant.

62. The plant cell of claim 61, further comprising a 5' UTR, the 5' UTR comprising a nucleic acid sequence selected from SEQ ID NO:43-79, wherein the nucleic acid sequence encoding the 5' UTR is operatively linked to a nucleic acid sequence encoding the promoter.

63. The plant cell according to claim 61, wherein the reporter gene is selected from genes encoding fluorescent proteins, genes encoding bioluminescent proteins, and genes encoding pigment proteins.

64. The plant cell of claim 63, wherein the reporter gene encodes a fluorescent protein.

65. The plant cell of claim 64, wherein the reporter gene encodes bFLO_GFP.

66. The plant cell according to claim 61, wherein the nucleic acid molecule is in soybean (soybean, Glycine max Gene regulatory activity is exhibited in plants.

67. The plant cell of claim 61, wherein the promoter comprises a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:

4.

68. The plant cell of claim 67, wherein the promoter is operatively linked to a 5' UTR comprising the nucleic acid sequence of SEQ ID NO:

46.

69. The plant cell of claim 67, wherein the promoter comprises SEQ ID NO:

4.

70. The plant cell of claim 67, wherein the promoter comprises SEQ ID NO: 4 and is operatively linked to a 5' UTR comprising SEQ ID NO:

46.

71. The plant cell of claim 67, wherein the reporter gene is selected from genes encoding fluorescent proteins, genes encoding bioluminescent proteins, and genes encoding pigment proteins.

72. The plant cell of claim 71, wherein the reporter gene encodes a fluorescent protein.

73. The plant cell of claim 72, wherein the reporter gene encodes bFLO_GFP.

74. The plant cell according to claim 67, wherein the nucleic acid molecule is in soybean (soybean, Glycine max Gene regulatory activity is exhibited in plants.

75. The plant cell of claim 61, wherein the promoter comprises a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO:

17.

76. The plant cell of claim 75, wherein the promoter is operatively linked to a 5' UTR comprising the nucleic acid sequence of SEQ ID NO:

59.

77. The plant cell of claim 75, wherein the promoter comprises SEQ ID NO:

17.

78. The plant cell of claim 75, wherein the promoter comprises SEQ ID NO: 17 and is operatively linked to a 5' UTR comprising SEQ ID NO:

59.

79. The plant cell of claim 75, wherein the reporter gene is selected from genes encoding fluorescent proteins, genes encoding bioluminescent proteins, and genes encoding pigment proteins.

80. The plant cell of claim 79, wherein the reporter gene encodes a fluorescent protein.

81. The plant cell of claim 80, wherein the reporter gene encodes bFLO_GFP.

82. The plant cell according to claim 75, wherein the nucleic acid molecule is in soybean (soybean, Glycine max Gene regulatory activity is exhibited in plants.

83. The plant cell of claim 61, wherein the promoter comprises a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO:

18.

84. The plant cell of claim 83, wherein the promoter is operatively linked to a 5' UTR comprising the nucleic acid sequence of SEQ ID NO:

60.

85. The plant cell of claim 83, wherein the promoter comprises SEQ ID NO:

18.

86. The plant cell of claim 83, wherein the promoter comprises SEQ ID NO: 18 and is operatively linked to a 5' UTR comprising SEQ ID NO:

60.

87. The plant cell of claim 83, wherein the reporter gene is selected from genes encoding fluorescent proteins, genes encoding bioluminescent proteins, and genes encoding pigment proteins.

88. The plant cell of claim 87, wherein the reporter gene encodes a fluorescent protein.

89. The plant cell of claim 88, wherein the reporter gene encodes bFLO_GFP.

90. The plant cell according to claim 83, wherein the nucleic acid molecule is in soybean (soybean, Glycine max Gene regulatory activity is exhibited in plants.

91. A plant comprising a nucleic acid molecule according to any one of claims 1-30, a binary carrier according to any one of claims 31-60, and / or a plant cell according to any one of claims 61-90.

92. The plant according to claim 91, wherein the plant is soybean (soybean, Glycine max )plant.

93. A seed comprising a nucleic acid molecule according to any one of claims 1-30, a binary carrier according to any one of claims 31-60, and / or a plant cell according to any one of claims 61-90.

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