SPATIO-TEMPORAL PROMOTERS FOR POLYNUCLEOTIDE EXPRESSION IN PLANTS
Provided herein are compositions and methods for expressing a polynucleotide of interest in a plant or plant part. Compositions include promoter molecules that initiate spatio-temporal expression of polynucleotides of interest, and DNA constructs comprising the promoter molecule operably linked to one or more polynucleotides of interest. Plants and plant parts comprising the compositions or produced according to the methods are also provided.
1 . A DNA construct comprising, in operable linkage:
(a) a first promoter molecule comprising a nucleic acid sequence that shares at least 80% sequence identity with any one of SEQ ID NOs: 1-27 and retains transcription initiation activity, or a nucleic acid sequence of any one of SEQ ID NOs: 1-27; and
(b) a first polynucleotide of interest.
2 . The DNA construct of claim 1 , wherein the first polynucleotide of interest encodes a morphogen, a transforming protein, a recombination modulator, a repair modulator, a defense pathway modulator, a nuclease, a selectable marker, and/or a regulatory RNA.
3 . The DNA construct of claim 1 or 2 , wherein said first polynucleotide of interest comprises a nucleic acid sequence that encodes: (i) a polypeptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 44 or SEQ ID NO: 45, wherein said polypeptide has morphogenic activity; or (ii) a polypeptide comprising an amino acid sequence of SEQ ID NO: 44 or SEQ ID NO: 45.
4 . The DNA construct of claim 2 , wherein the regulatory RNA is one or more of a microRNA (miRNA), a short-hairpin RNA, a guide RNA, a transposase, a homology-directed repair enhancer, and a non-homologous end-joining suppressor.
5 . The DNA construct of any one of claims 1-4 , wherein the promoter molecule further comprises a 5′UTR sequence, a 5′UTR intron sequence, an exon sequence from a coding region, and/or an intron sequence from a coding region of a plant genome.
6 . The DNA construct of any one of claims 1-5 , further comprising a second promoter molecule operably linked to a second polynucleotide of interest.
7 . The DNA construct of claim 6 , wherein the first and second polynucleotides of interest each encode a morphogen.
8 . The DNA construct of claim 7 , wherein the first polynucleotide of interest operably linked to the first promoter molecule comprises a nucleic acid sequence that encodes: (i) a polypeptide comprising an amino acid sequence that shares at least 80% sequence identity with SEQ ID NO: 44, wherein said polypeptide has morphogenic activity, or (ii) a polypeptide comprising an amino acid sequence of SEQ ID NO: 44; and
wherein the second polynucleotide of interest operably linked to the second promoter molecule comprises a nucleic acid sequence that encodes: (iii) a polypeptide comprising an amino acid sequence that shares at least 80% sequence identity with SEQ ID NO: 45, wherein said polypeptide has morphogenic activity, or (iv) a polypeptide comprising an amino acid sequence of SEQ ID NO: 45.
9 . The DNA construct of claim 7 , wherein the first polynucleotide of interest operably linked to the first promoter molecule comprises a nucleic acid sequence that encodes: (i) a polypeptide comprising an amino acid sequence that shares at least 80% sequence identity with SEQ ID NO: 45, wherein said polypeptide has morphogenic activity, or (ii) a polypeptide comprising an amino acid sequence of SEQ ID NO: 45; and
wherein the second polynucleotide of interest operably linked to the second promoter molecule comprises a nucleic acid sequence that encodes: (iii) a polypeptide comprising an amino acid sequence that shares at least 80% sequence identity with SEQ ID NO: 44, wherein said polypeptide has morphogenic activity, or (iv) a polypeptide comprising an amino acid sequence of SEQ ID NO: 44.
10 . The DNA construct of any one of claims 6-9 , wherein the first promoter molecule comprises a nucleic acid sequence that shares at least 80% sequence identity with any one of SEQ ID NOs: 1-3 and retains transcription initiation activity, or a nucleic acid sequence of any one of SEQ ID NOs: 1-3.
11 . The DNA construct of any one of claims 6-10 , wherein the second promoter molecule comprises a nucleic acid sequence that shares at least 80% sequence identity with any one of SEQ ID NOs: 1-27 and retains transcription initiation activity, or a nucleic acid sequence of any one of SEQ ID NOs: 1-27.
12 . The DNA construct of any one of claims 1-11 , wherein said promoter molecule(s) comprising a nucleic acid sequence that shares at least 80% sequence identity with any one of SEQ ID NOs: 1-27 and retains transcription initiation activity, or a nucleic acid sequence of any one of SEQ ID NOs: 1-27, initiates expression of the operably linked polynucleotide of interest limited to a seed-to-seedling developmental phase when the DNA construct is introduced in a plant or plant part.
13 . The DNA construct of any one of claims 1-12 , wherein said promoter molecule(s) comprising a nucleic acid sequence that shares at least 80% sequence identity with any one of SEQ ID NOs: 1-27 and retains transcription initiation activity, or a nucleic acid sequence of any one of SEQ ID NOs: 1-27, initiates embryonic tissue-preferred expression of the operably linked polynucleotide of interest when the DNA construct is introduced in a plant or plant part.
14 . The DNA construct of claim 13 , wherein the preferred embryonic tissue is epicotyl, hypocotyl, radicle, cotyledon, or a combination thereof.
15 . A cell comprising the DNA construct of any one of claims 1-14 .
16 . The cell of claim 15 , wherein the cell is a plant cell.
17 . A plant or plant part comprising the DNA construct of any one of claims 1-14 or the cell of claim 15 or 16 .
18 . The plant or plant part of claim 17 , wherein said plant is selected from the group consisting of corn ( Zea mays ), Brassica species, Brassica napus, Brassica rapa, Brassica juncea , alfalfa ( Medicago sativa ), pea ( Pisum sativum ), fava bean ( Vicia faba ), common bean ( Phaseolus vulgaris ), chickpea ( Cicer arietinum ), mung bean ( Vigna radiata ), white lupin ( Lupinus albus ), rice ( Oryza sativa ), rye ( Secale cereale ), sorghum ( Sorghum bicolor, Sorghum vulgare ), millet, pearl millet ( Pennisetum glaucum ), proso millet ( Panicum miliaceum ), foxtail millet ( Setaria italica ), finger millet ( Eleusine coracana ), sunflower ( Helianthus annuus ), safflower ( Carthamus tinctorius ), wheat ( Triticum aestivum ), soybean ( Glycine max ), tobacco ( Nicotiana tabacum ), potato ( Solanum tuberosum ), peanuts ( Arachis hypogaea ), cotton ( Gossypium barbadense, Gossypium hirsutum ), sweet potato ( Ipomoea batatus ), cassava ( Manihot esculenta ), coffee ( Coffea spp.), coconut ( Cocos nucifera ), pineapple ( Ananas comosus ), citrus trees ( Citrus spp.), cocoa ( Theobroma cacao ), tea ( Camellia sinensis ), banana ( Musa spp.), avocado ( Persea americana ), fig ( Ficus casica ), guava ( Psidium guajava ), mango ( Mangifera indica ), olive ( Olea europaea ), papaya ( Carica papaya ), cashew ( Anacardium occidentale ), macadamia ( Macadamia integrifolia ), almond ( Prunus amygdalus ), sugar beets ( Beta vulgaris ), sugarcane ( Saccharum spp.), oats, barley, vegetables, ornamentals, and conifers.
19 . The plant or plant part of claim 17 or 18 , wherein said plant is a dicot.
20 . The plant or plant part of claim 19 , wherein said plant is a legume.
21 . A method of expressing a polynucleotide of interest in a plant or plant part comprising introducing a DNA construct into said plant or plant part, wherein the DNA construct comprises, in operable linkage:
(a) a first promoter molecule comprising a nucleic acid sequence that shares at least 80% sequence identity with any one of SEQ ID NOs: 1-27 and retains transcription initiation activity, or a nucleic acid sequence of any one of SEQ ID NOs: 1-27; and
(b) a first polynucleotide of interest.
22 . The method of claim 21 , said method comprising:
introducing a DNA construct into a plant cell, wherein the DNA construct comprises, in operable linkage: (a) a first promoter molecule comprising a nucleic acid sequence that shares at least 80% sequence identity with any one of SEQ ID NOs: 1-27 and retains transcription initiation activity, or a nucleic acid sequence of any one of SEQ ID NOs: 1-27; and (b) a first polynucleotide of interest; and
regenerating a transformed plant or plant part from said plant cell.
23 . The method of claim 22 , wherein the method increases normal shoot formation, frequency of shoot producing plants or plant parts, and/or number of regenerated shoots from transformed plants or plant parts relative to a control method comprising introducing a control DNA construct comprising a control promoter molecule into a plant cell.
24 . The method of claim 23 , wherein the frequency of shoot producing plants or plant parts is increased by about 10% to about 500% relative to a control method.
25 . The method of claim 23 , wherein the number of regenerated shoots from transformed plants or plant parts is increased by about 10% to about 1200% relative to a control method.
26 . The method of any one of claims 21-25 , wherein the first polynucleotide of interest encodes a morphogen, a transforming protein, a recombination modulator, a repair modulator, a defense pathway modulator, a nuclease, a selectable marker, and/or a regulatory RNA.
27 . The method of claim 26 , wherein said first polynucleotide of interest comprises a nucleic acid sequence that encodes: (i) a polypeptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 44 or SEQ ID NO: 45, wherein said polypeptide has morphogenic activity; or (ii) a polypeptide comprising an amino acid sequence of SEQ ID NO: 44 or SEQ ID NO: 45.
28 . The method of claim 26 , wherein the regulatory RNA is one or more of a microRNA (miRNA), a short-hairpin RNA, a guide RNA, a transposase, a homology-directed repair enhancer, and a non-homologous end-joining suppressor.
29 . The method of any one of claims 21-28 , wherein the first promoter molecule further comprises a 5′UTR sequence, a 5′UTR intron sequence, an exon sequence from a coding region, and/or an intron sequence from a coding region of a plant genome.
30 . The method of any one of claims 21-29 , wherein the DNA construct further comprises a second promoter molecule operably linked to a second polynucleotide of interest.
31 . The method of any one of claims 21-29 , further comprising introducing a second DNA construct into said plant or plant part, wherein the second DNA construct comprises a second promoter molecule operably linked to a second polynucleotide of interest.
32 . The method of claim 30 or 31 , wherein the first and second polynucleotides of interest each encode a morphogen.
33 . The method of claim 32 , wherein the first polynucleotide of interest operably linked to the first promoter molecule comprises a nucleic acid sequence that encodes: (i) a polypeptide comprising an amino acid sequence that shares at least 80% sequence identity with SEQ ID NO: 44, wherein said polypeptide has morphogenic activity, or (ii) a polypeptide comprising an amino acid sequence of SEQ ID NO: 44; and
wherein the second polynucleotide of interest operably linked to the second promoter molecule comprises a nucleic acid sequence that encodes: (iii) a polypeptide comprising an amino acid sequence that shares at least 80% sequence identity with SEQ ID NO: 45, wherein said polypeptide has morphogenic activity, or (iv) a polypeptide comprising an amino acid sequence of SEQ ID NO: 45.
34 . The method of claim 32 , wherein the first polynucleotide of interest of the first DNA construct comprises a nucleic acid sequence that encodes: (i) a polypeptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 45, wherein said polypeptide has morphogenic activity; or (ii) a polypeptide comprising an amino acid sequence of SEQ ID NO: 45; and
wherein the second polynucleotide of interest of the second DNA construct comprises a nucleic acid sequence that encodes: (iii) a polypeptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 44, wherein said polypeptide has morphogenic activity; or (iv) a polypeptide comprising an amino acid sequence of SEQ ID NO: 44.
35 . The method of any one of claims 21-34 , wherein the first promoter molecule comprises a nucleic acid sequence that shares at least 80% sequence identity with any one of SEQ ID NOs: 1-3 and retains transcription initiation activity, or a nucleic acid sequence of any one of SEQ ID NOs: 1-3.
36 . The method of any one of claims 30-35 , wherein the second promoter molecule comprises a nucleic acid sequence that shares at least 80% sequence identity with any one of SEQ ID NOs: 1-27 and retains transcription initiation activity, or a nucleic acid sequence of any one of SEQ ID NOs: 1-27.
37 . The method of any one of claims 21-36 , wherein said promoter molecule(s) comprising a nucleic acid sequence that shares at least 80% sequence identity with any one of SEQ ID NOs: 1-27 and retains transcription initiation activity, or a nucleic acid sequence of any one of SEQ ID NOs: 1-27, initiates expression of the operably linked polynucleotide of interest limited to a seed-to-seedling developmental phase in the plant or plant part.
38 . The method of any one of claims 21-37 , wherein the said promoter molecule(s) comprising a nucleic acid sequence that shares at least 80% sequence identity with any one of SEQ ID NOs: 1-27 and retains transcription initiation activity, or a nucleic acid sequence of any one of SEQ ID NOs: 1-27, initiates embryonic tissue-preferred expression of the operably linked polynucleotide of interest in the plant or plant part.
39 . The method of claim 38 , wherein the preferred embryonic tissue is epicotyl, hypocotyl, radicle, cotyledon, or a combination thereof.
40 . The method of any one of claims 21-39 , wherein said plant is selected from the group consisting of corn ( Zea mays ), Brassica species, Brassica napus, Brassica rapa, Brassica juncea , alfalfa ( Medicago sativa ), pea ( Pisum sativum ), fava bean ( Vicia faba ), common bean ( Phaseolus vulgaris ), chickpea ( Cicer arietinum ), mung bean ( Vigna radiata ), white lupin ( Lupinus albus ), rice ( Oryza sativa ), rye ( Secale cereale ), sorghum ( Sorghum bicolor, Sorghum vulgare ), millet, pearl millet ( Pennisetum glaucum ), proso millet ( Panicum miliaceum ), foxtail millet ( Setaria italica ), finger millet ( Eleusine coracana ), sunflower ( Helianthus annuus ), safflower ( Carthamus tinctorius ), wheat ( Triticum aestivum ), soybean ( Glycine max ), tobacco ( Nicotiana tabacum ), potato ( Solanum tuberosum ), peanuts ( Arachis hypogaea ), cotton ( Gossypium barbadense, Gossypium hirsutum ), sweet potato ( Ipomoea batatus ), cassava ( Manihot esculenta ), coffee ( Coffea spp.), coconut ( Cocos nucifera ), pineapple ( Ananas comosus ), citrus trees ( Citrus spp.), cocoa ( Theobroma cacao ), tea ( Camellia sinensis ), banana ( Musa spp.), avocado ( Persea americana ), fig ( Ficus casica ), guava ( Psidium guajava ), mango ( Mangifera indica ), olive ( Olea europaea ), papaya ( Carica papaya ), cashew ( Anacardium occidentale ), macadamia ( Macadamia integrifolia ), almond ( Prunus amygdalus ), sugar beets ( Beta vulgaris ), sugarcane ( Saccharum spp.), oats, barley, vegetables, ornamentals, and conifers.
41 . The method of any one of claims 21-40 , wherein said plant is a dicot.
42 . The method of claim 41 , wherein said plant is a legume.
43 . The method of any one of claims 21-42 , wherein the polynucleotide(s) of interest is stably inserted into a genome of said plant or plant part.
44 . A plant or plant part produced by the method of any one of claims 21-43 , wherein said plant or plant part comprises said DNA construct.