Controlling gene expression in plants using fusion protein containing LexA binding domain and DREB transactivation domain
The present invention relates to the regulation of transgene expression in plants through a transactivation system which comprises the following: (1) a promoter comprising LexA binding sites; and (2) a fusion transactivator protein comprising a LexA DNA-binding domain and an activation domain, such as the transactivation domain of a C-repeat binding factor protein, for example, from a dehydration responsive element (DREB) factor.
1. A plant cell comprising:
(a) a heterologous polynucleotide operably linked to a minimal promoter and a hybrid LexA operator sequence, wherein the hybrid LexA operator sequence comprises one copy each of at least two non-identical LexA operator sequences, wherein the hybrid LexA operator sequence comprises at least one sequence selected from the group consisting of: the polynucleotide of SEQ ID NO:22 and the polynucleotide of SEQ ID NO:23; and
(b) a recombinant DNA construct comprising a promoter functional in a plant cell operably linked to a polynucleotide encoding a fusion protein, wherein the polynucleotide comprises:
(1) a first nucleotide sequence encoding a first amino acid sequence having a DNA-binding domain wherein the first amino acid sequence is SEQ ID NO:36 or a functional fragment of SEQ ID NO: 36; and
(2) a second nucleotide sequence encoding a second amino acid sequence having a transactivation domain wherein the second amino acid sequence is SEQ ID NO: 90 or a functional fragment of SEQ ID NO: 90, wherein the transactivation domain is operably linked to a nuclear localization sequence (NLS);
wherein the fusion protein is capable of activating transcription of a heterologous polynucleotide that is operably linked to the hybrid LexA operator sequence.
2. The recombinant DNA construct of claim 1 , wherein the heterologous polynucleotide encodes a heterologous polypeptide capable of modifying an agronomic characteristic in the plant cell.
3. The plant cell of claim 1 , wherein the NLS in the recombinant DNA construct comprises the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:39.
4. The plant cell of claim 1 , wherein the promoter in the recombinant DNA construct is a tissue-specific promoter.
5. The plant cell of claim 1 , wherein the promoter in the recombinant DNA construct is an inducible promoter.
6. A plant obtained from the plant cell of claim 1 .
7. A seed produced by the plant of claim 6 , wherein said seed comprises said DNA construct and said heterologous polynucleotide.
8. A plant obtained from the plant cell of claim 4 .
9. A seed produced by the plant of claim 8 , wherein said seed comprises said DNA construct and said heterologous polynucleotide.
10. A method of regulating the expression of a heterologous polynucleotide in a plant, wherein the method comprises the steps of:
a. obtaining a plant comprising: (i) a first recombinant DNA construct, where the first recombinant DNA construct is the recombinant DNA construct of claim 1 ; and (ii) a second recombinant DNA construct comprising a heterologous polynucleotide operably linked to a minimal promoter and at least one LexA operator sequence, wherein the heterologous polynucleotide encodes a heterologous polypeptide capable of modifying an agronomic characteristic in the plant cell; and
b. growing the plant of step (a) under conditions in which the first recombinant DNA construct is expressed.
11. A method of regulating the expression of a heterologous polynucleotide in a plant, wherein the method comprises the steps of:
a. obtaining a plant comprising a first recombinant DNA construct, where the first recombinant DNA construct is the recombinant DNA construct of claim 1 ;
b. introducing into the plant of step (a) a second recombinant DNA construct comprising a heterologous polynucleotide operably linked to a minimal promoter and at least one LexA operator sequence, wherein the heterologous polynucleotide encodes a heterologous polypeptide capable of modifying an agronomic characteristic in the plant cell; and
c. growing the plant of step (b) under conditions in which the first recombinant DNA construct is expressed.
12. A method of regulating the expression of a heterologous polynucleotide in a plant, wherein the method comprises the steps of:
a. introducing into a regenerable plant cell a first recombinant DNA construct, where the first recombinant DNA construct is the recombinant DNA construct of claim 1 , and a second recombinant DNA construct comprising a heterologous polynucleotide operably linked to a minimal promoter and at least one LexA operator sequence, wherein the heterologous polynucleotide encodes a heterologous polypeptide capable of modifying an agronomic characteristic in the plant cell;
b. regenerating a transgenic plant from the regenerable plant cell of step (a) wherein the transgenic plant comprises in its genome the first recombinant DNA construct and the second recombinant DNA construct; and
c. growing the transgenic plant of step (b) under conditions in which the first recombinant DNA construct is expressed.
13. A method of regulating the expression of a heterologous polynucleotide in a plant, wherein the method comprises the steps of:
a. introducing into a regenerable plant cell a first recombinant DNA construct, where the first recombinant DNA construct is the recombinant DNA construct of claim 1 , and a second recombinant DNA construct comprising a heterologous polynucleotide operably linked to a minimal promoter and at least one LexA operator sequence, wherein the heterologous polynucleotide encodes a heterologous polypeptide capable of modifying an agronomic characteristic in the plant cell;
b. regenerating a transgenic plant from the regenerable plant cell of step (a) wherein the transgenic plant comprises in its genome the first recombinant DNA construct and the second recombinant DNA construct;
c. obtaining a progeny plant derived from the transgenic plant of step (b), wherein the progeny plant comprises in its genome the first recombinant DNA construct and the second recombinant DNA construct; and
d. growing the progeny plant of step (c) under conditions in which the first recombinant DNA construct is expressed.
14. The plant of claim 6 , wherein the hybrid LexA operator sequence comprises at least one sequence of the polynucleotide of SEQ ID NO:22 and at least one sequence of the polynucleotide of SEQ ID NO:23.
15. The plant of claim 14 , wherein the hybrid LexA operator sequence comprises more than one copy of the polynucleotide sequence of SEQ ID NO:22 or the polynucleotide sequence of SEQ ID NO:23.
16. The plant cell of claim 1 , wherein the hybrid LexA operator sequence comprises at least one sequence of the polynucleotide of SEQ ID NO:22 and at least one sequence of the polynucleotide of SEQ ID NO:23.
17. The plant cell of claim 1 , wherein the hybrid LexA operator sequence comprises more than one copy of the polynucleotide sequence of SEQ ID NO:22 or the polynucleotide sequence of SEQ ID NO:23.