Synthetic ethylene-responsive binding element and uses thereof
Provided is a recombinant nucleic acid construct comprising a sequence of Formula I (SEQ ID NO:48): X—N 1 —N 2 —N 3 —N 4 —N 5 —N 6 —N 7 —N 8 —N 9 —N 10 —N 11 —Y as an ethylene binding sequence (EBS). Tandems of two or more directly adjoining EBS are also described, and may be included in an expression cassette useful for modulating the expression of a nucleic acid of interest in a plant in response to plant hormone ethylene.
1. A recombinant nucleic acid construct comprising two or more directly adjoining ethylene binding sequences (EBS), wherein each of the EBS is a sequence of Formula I (SEQ ID NO:48):
X—N 1 —N 2 —N 3 —N 4 —N 5 —N 6 —N 7 —N 8 —N 9 —N 10 —N 11 —Y (I)
wherein:
N 1 is A or G;
N 2 is T;
N 3 is G, T or A;
N 4 is C;
N 5 is A;
N 6 is A or T;
N 7 is T;
N 8 is G;
N 9 is A, T or C;
N 10 is A;
N 11 is T or C; and
X and Y are each independently present or absent and when present is each independently a spacer sequence comprising 1 to 50 nucleotides.
2. The recombinant nucleic acid construct of claim 1 , wherein the two or more EBS of Formula I comprise two or more of SEQ ID NOS:1-11.
3. The recombinant nucleic acid construct of claim 1 , wherein the two or more EBS of Formula I are operably linked to a core promoter, optionally wherein the core promoter is within 0 to 2,000 base pairs downstream (3′) of the two or more EBS of Formula I.
4. The recombinant nucleic acid construct of claim 1 , wherein the two or more EBS of Formula I comprise EBS which are not identical with one another.
5. The recombinant nucleic acid construct of claim 1 , wherein the two or more EBS of Formula I comprise four or five EBS of Formula I.
6. The recombinant nucleic acid construct of claim 1 , wherein the two or more EBS of Formula I comprise six, seven, eight, or nine EBS of Formula I.
7. The recombinant nucleic acid construct of claim 1 , wherein the two or more EBS of Formula I comprise ten, eleven or twelve EBS of Formula I, and optionally up to 50, 75 or 100 EBS of Formula I.
8. The recombinant nucleic acid construct of claim 1 , wherein the EBS of Formula I does not comprise a transcription factor binding element that is not an EBS of Formula I.
9. The recombinant nucleic acid construct of claim 8 , wherein the construct comprises a sequence selected from the group consisting of SEQ ID NOS:14 and 27-46; and sequences with 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity thereto.
10. An expression cassette comprising the recombinant nucleic acid construct of claim 1 operably linked to a core promotor, and optionally one or more of a subcellular localization signal, a nucleic acid of interest, and a transcriptional terminator.
11. The expression cassette of claim 10 , wherein the nucleic acid of interest is present and encodes a reporter protein.
12. The expression cassette of claim 10 , wherein the nucleic acid of interest is present and encodes an antioxidant protein, a toxin, a vitamin-biosynthesis protein, a pigment protein, a pathogen defense protein, or a flavor inducing enzyme.
13. The expression cassette of claim 10 , wherein the expression cassette comprises, from 5′ to 3′, the recombinant nucleic acid construct, the minimal promotor, the nucleic acid of interest, and the transcriptional terminator.
14. A plant cell comprising the expression cassette of claim 10 .
15. A plant comprising the plant cell of claim 14 .
16. The plant of claim 15 , wherein the plant is Arabidopsis , tomato, tobacco, maize, or rice.
17. A method of modulating the expression of a nucleic acid of interest in a plant in response to ethylene, the method comprising
introducing into a plant cell the expression cassette of claim 10 to produce a transformed plant cell;
regenerating a transformed plant from the transformed plant cell; and
exposing the transformed plant, or a plant part or plant cell therefrom, to the ethylene, whereby the nucleic acid of interest is expressed.
18. The method of claim 17 , wherein the recombinant nucleic acid construct and the nucleic acid of interest are integrated into the genome of the plant.
19. The method of claim 17 , wherein the recombinant nucleic acid construct and the nucleic acid of interest are not integrated into the genome of the plant.
20. The method of claim 17 , wherein the exposing is carried out by applying ethylene or an ethylene precursor to the transformed plant, or plant part or plant cell therefrom.