PROMOTER ELEMENTS FOR IMPROVED POLYNUCLEOTIDE EXPRESSION IN PLANTS
Provided herein are compositions and methods for expressing a polynucleotide of interest in a plant or plant part. Compositions include nucleic acid molecules comprising a promoter sequence, and DNA constructs comprising the promoter molecule operably linked to one or more polynucleotides of interest. Plants and plant parts comprising the compositions or regenerated according to the methods are also provided.
1 . A nucleic acid molecule comprising a promoter sequence, wherein the promoter sequence comprises one or more deletions, substitutions, and/or insertions relative to the nucleic acid sequence of any one of SEQ ID NOs: 1-4, 9-39, and 43-46; shares at least 75% sequence identity with any one of SEQ ID NOs: 1-4 and 9-39, and 43-46; and retains transcription initiation function.
2 . The nucleic acid molecule of claim 1 :
wherein said one or more deletions, substitutions, and/or insertions are located in a 3′ region of said promoter sequence; or
wherein the promoter sequence shares at least 80% sequence identity with any one of SEQ ID NOs: 5-8, or comprises the nucleic acid sequence of any one of SEQ ID NOs: 5-8, and wherein the promoter sequence retains transcription initiation function; or
wherein the nucleic acid molecule further comprises a 5′ untranslated region (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.
3 - 4 . (canceled)
5 . A DNA construct comprising, in operable linkage:
a. the nucleic acid molecule of claim 1 , or a promoter molecule comprising a nucleic acid sequence that shares at least 80% sequence identity with any one of SEQ ID NOs: 1-4, 9-39, and 43-46, and retains transcription initiation function or a promoter molecule comprising a nucleic acid sequence of any one of SEQ ID NOs: 1-4, 9-39, and 43-46; and
b. a polynucleotide of interest.
6 . The DNA construct of claim 5 :
wherein the DNA construct comprises, in operable linkage:
a. a promoter molecule comprising a nucleic acid sequence that shares at least 80% sequence identity with any one of SEQ ID NOs: 1-4, and retains transcription initiation function, or a promoter molecule comprising a nucleic acid sequence of any one of SEQ ID NOs: 1-4; and
b. a polynucleotide of interest, or
wherein the promoter molecule further comprises a 5′ untranslated region (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.
7 . (canceled)
8 . The DNA construct of claim 5 , wherein the polynucleotide of interest encodes a guide RNA and/or a nuclease.
9 . The DNA construct of claim 8 :
(i) wherein the polynucleotide of interest encodes a guide RNA, and wherein the DNA construct further comprises, in operable linkage:
a. the nucleic acid molecule of any one of claims 1 - 4 ; a promoter molecule comprising a nucleic acid sequence that has at least 80% sequence identity to any one of SEQ ID NOs: 1-4, 9-39, 43-52, and 56-61, and retains transcription initiation function; or a promoter molecule comprising a nucleic acid sequence of any one of SEQ ID NOs: 1-4, 9-39, 43-52, and 56-61; and
b. a polynucleotide of interest encoding a nuclease; or
(ii) wherein the polynucleotide of interest encodes a nuclease, and wherein the DNA construct further comprises, in operable linkage:
c. the nucleic acid molecule of any one of claims 1 - 4 ; a promoter molecule comprising a nucleic acid sequence that has at least 80% sequence identity to any one of SEQ ID NOs: 1-4, 47-49, 51, and 53, and retains transcription initiation function; or a promoter molecule comprising a nucleic acid sequence of any one of SEQ ID NOs: 1-4, 47-49, 51, and 53; and
d. a polynucleotide of interest encoding a guide RNA.
10 - 13 . (canceled)
14 . The DNA construct of claim 8 , wherein the nuclease is a clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated Cas endonuclease, or the nuclease is further operably linked to one or more nuclear localization sequences (NLSs) and/or one or more epitope tags.
15 . The DNA construct of claim 5 , comprising a nucleic acid molecule encoding a selectable marker, morphogen gene, reporter gene, and/or a regulatory RNA, operably linked to a promoter molecule.
16 . The DNA construct of claim 15 , wherein the promoter molecule operably linked to the regulatory RNA comprises: a nucleic acid molecule comprising one or more deletions, substitutions, and/or insertions relative to the nucleic acid sequence of any one of SEQ ID NOs: 1-4, 9-39, and 43-46, shares at least 75% sequence identity with any one of SEQ ID NOs: 1-4 and 9-39, and 43-46, and retains transcription initiation function; a nucleic acid sequence that has at least 80% sequence identity to any one of SEQ ID NOs: 1-4, 47-49, 51, and 53, and retains transcription initiation function; or a nucleic acid sequence of any one of SEQ ID NOs: 1-4, 47-49, 51, and 53.
17 . A cell comprising the nucleic acid molecule of claim 1 or a DNA construct comprising, in operable linkage, said nucleic acid molecule of claim 1 and a polynucleotide of interest.
18 - 19 . (canceled)
20 . A plant or plant part comprising, in operable linkage:
a. a nucleic acid molecule comprising a promoter sequence, wherein the promoter sequence comprises one or more deletions, substitutions, and/or insertions relative to the nucleic acid sequence of any one of SEQ ID NOs: 1-4, 9-39, and 43-46; shares at least 75% sequence identity with any one of SEQ ID NOs: 1-4, 9-39, and 43-46; and retains transcription initiation function;
a promoter molecule comprising a nucleic acid sequence that has at least 80% sequence identity to any one of SEQ ID NOs: 1-4, 9-39, and 43-46, and retains transcription initiation function; or
a promoter molecule comprising a nucleic acid sequence of any one of SEQ ID NOs: 1-4, 9-39, and 43-46; and
b. a polynucleotide of interest.
21 . The plant or plant part of claim 20 :
wherein said one or more deletions, substitutions, and/or insertions are located in a 3′ region of said promoter sequence; or
wherein the promoter sequence shares at least 80% sequence identity with any one of SEQ ID NOs: 5-8, or comprises the nucleic acid sequence of any one of SEQ ID NOs: 5-8, and retains transcription initiation function; or
wherein the nucleic acid molecule comprising a promoter sequence or the promoter molecule comprises a 5′ untranslated region (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; or
wherein the nucleic acid molecule, the DNA construct, or part thereof is stably inserted in the genome of said plant or plant part; or
wherein said plant or plant part is a legume.
22 - 26 . (canceled)
27 . A method of expressing a polynucleotide of interest in a plant or plant part comprising introducing a DNA construct into the plant or plant part, wherein the DNA construct comprises, in operable linkage:
a. a nucleic acid molecule comprising a promoter sequence, wherein the promoter sequence comprises one or more deletions, substitutions, and/or insertions relative to the nucleic acid sequence of any one of SEQ ID NOs: 1-4, 9-39, and 43-46; shares at least 75% sequence identity with any one of SEQ ID NOs: 1-4, 9-39, and 43-46; and retains transcription initiation function;
a promoter molecule comprising a nucleic acid sequence that has at least 80% sequence identity to any one of SEQ ID NOs: 1-4, 9-39, and 43-46, and retains transcription initiation function; or
a promoter molecule comprising a nucleic acid sequence of any one of SEQ ID NOs: 1-4, 9-39, and 43-46; and
b. a polynucleotide of interest.
28 . The method of claim 27 , comprising:
introducing the DNA construct into a plant cell; and
regenerating a transformed plant or plant part from said plant cell.
29 . The method of claim 27 :
wherein said one or more deletions, substitutions, and/or insertions are located in a 3′ region of said promoter sequence; or
wherein the promoter sequence shares at least 80% sequence identity with any one of SEQ ID NOs: 5-8, or comprises the nucleic acid sequence of any one of SEQ ID NOs: 5-8, and retains transcription initiation function; or
wherein the nucleic acid molecule comprising the promoter sequence or the promoter molecule further comprises a 5′ untranslated region (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 - 32 . (canceled)
33 . The method of claim 27 , wherein the polynucleotide of interest encodes a guide RNA and/or a nuclease.
34 . The method of claim 27 :
(i) wherein the polynucleotide of interest encodes a guide RNA, and wherein the DNA construct further comprises, in operable linkage:
a. the nucleic acid molecule of any one of claims 1 - 4 ; a promoter molecule comprising a nucleic acid sequence that has at least 80% sequence identity to any one of SEQ ID NOs: 1-4, 9-39, 43-52, and 56-61, and retains transcription initiation function; or a promoter molecule comprising a nucleic acid sequence of any one of SEQ ID NOs: 1-4, 9-39, 43-52, and 56-61; and
b. a polynucleotide of interest encoding a nuclease; or
(ii) wherein the polynucleotide of interest encodes a nuclease, and the DNA construct further comprises, in operable linkage:
a. the nucleic acid molecule of any one of claims 1 - 4 ; a promoter molecule comprising a nucleic acid sequence that has at least 80% sequence identity to any one of SEQ ID NOs: 1-4, 47-49, 51, and 53, and retains transcription initiation function; or a promoter molecule comprising a nucleic acid sequence of any one of SEQ ID NOs: 1-4, 47-49, 51, and 53; and
b. a polynucleotide of interest encoding a guide RNA; or
(iii) wherein the polynucleotide of interest encodes a nuclease, and the nuclease is further operably linked to one or more nuclear localization sequences (NLSs) and/or one or more epitope tags; or
(iv) wherein the DNA construct further comprises a nucleic acid molecule encoding a selectable marker and/or a regulatory RNA, operably linked to a promoter molecule; or
(v) wherein the nucleic acid molecule, the DNA construct, or part thereof is stably inserted in the genome of said plant or plant part.
35 - 42 . (canceled)
43 . The method of claim 27 :
wherein expression or function of one or more molecules encoded by the polynucleotide(s) of interest is increased in the plant or plant part relative to a control plant or plant part comprising the polynucleotide(s) of interest operably linked to a control promoter that does not comprise the promoter molecule of claim 27 , or
wherein the polynucleotide(s) of interest encodes a guide RNA and/or a nuclease, and wherein an efficiency of introducing a mutation to a genome of the plant or plant part is increased relative to the control plant or plant part; or
wherein the polynucleotide(s) of interest encodes a guide RNA and/or a nuclease, and wherein an efficiency of introducing a heritable mutation to a genome of a stably transformed and regenerated plant or plant part consistently across plant tissue is increased relative to the control plant or plant part.
44 - 46 . (canceled)
47 . The method of claim 27 , wherein said plant or plant part is a legume.
48 . A plant or plant part produced by the method of claim 27 , wherein said plant or plant part comprises said DNA construct or part thereof.