DOMINANT NEGATIVE MUTANT KRP PROTEIN PROTECTION OF ACTIVE CYCLIN-CDK COMPLEX INHIBITION BY WILD-TYPE KRP
Disclosed are mutant CDK inhibitor (CKI) polypeptides having dominant negative antagonist activity against wild-type CKI proteins, as well as related compositions, including nucleic acids and vectors encoding the mutant CKI polypeptides and transformed host cells and transgenic plants comprising such nucleic acids and vectors. Also disclosed are related methods for using the mutant proteins to modulate cell division in cells, particularly plant cells.
1 . A recombinant nucleic acid encoding a plant mutant CDK inhibitor (CKI) polypeptide,
wherein the mutant plant CKI polypeptide comprises: a CKI amino acid sequence having at least two modifications relative to a wild-type plant CKI polypeptide,
said wild-type CKI polypeptide comprising (a) a cyclin binding region conferring binding affinity for a cyclin and (b) a cyclin dependent kinase (CDK) binding region conferring binding affinity for a CDK;
wherein the wild-type CKI polypeptide binds to a cyclin/CDK complex;
wherein the at least two modifications are within the CDK binding region;
wherein the mutant CKI polypeptide can compete with said wild-type CKI for binding to the cyclin/CDK complex;
wherein said wild-type plant CKI polypeptide is a kinase inhibitor protein (KIP) related protein (KRP) family member and wherein said mutant plant CKI polypeptide does not inhibit kinase activity of the cyclin/CDK complex;
wherein said wild-type plant CKI polypeptide is from Canola, soy or corn; and
wherein said at least two modifications are within the region corresponding to amino acids 145-168 of Brassica KRP1 (BnKRP1) polypeptide encoded by the nucleotide sequence of SEQ ID NO: 23, wherein each of the at least two modifications is an amino acid substitution at:
(a) a position corresponding to amino acid 151 of BnKRP1; and
(b) a position corresponding to amino acid 153 of BnKRP1,
wherein the position corresponding to amino acid 151 of BnKRP1 is phenylalanine or tyrosine,
and wherein the position corresponding to amino acid 153 of BnKRP1 is phenylalanine, proline, isoleucine or valine.
2 . A vector comprising a replicon and the recombinant nucleic acid of claim 1 .
3 . The vector of claim 2 , which is an expression vector further comprising a promoter region operably linked to the recombinant nucleic acid.
4 . The expression vector of claim 3 , wherein the promoter is a constitutive promoter, an inducible promoter, a tissue-specific promoter, developmentally regulated promoter or a stage-specific promoter.
5 . The expression vector of claim 4 , wherein the constitutive promoter is a CaMV 35S promoter, the tissue-specific promoter is a seed specific promoter, and the stage-specific promoter is an embryo specific promoter.
6 . A host cell comprising the recombinant nucleic acid of claim 1 .
7 . A method of producing a mutant CKI polypeptide, said method comprising: culturing the host cell of claim 6 under conditions suitable for expression of the nucleic acid encoding the mutant CKI polypeptide.
8 . A transgenic plant comprising the recombinant nucleic acid of claim 1 , wherein the transgenic plant is Canola, soy or corn.
9 . A method of producing the transgenic plant of claim 8 , comprising: introducing into a plant a vector comprising the recombinant nucleic acid of claim 1 .
10 . A method of modulating cell division in a plant cell expressing a wild-type CKI polypeptide, said wild-type CKI polypeptide comprising (a) a cyclin binding region conferring binding affinity for a cyclin and (b) a CDK binding region conferring binding affinity for a CDK, the method comprising: expressing within the cell a mutant CKI polypeptide encoded by the recombinant nucleic acid of claim 1 , wherein the plant cell is from Canola, soy or corn.
11 . A method for increasing plant vigor, root mass, fruit size, plant seed size, plant seed number, plant yield, plant biomass, early germination, or cell division in a plant compared to a wild-type plant, comprising: expressing within the plant a mutant KRP polypeptide encoded by the recombinant nucleic acid of claim 1 , wherein the plant is Canola, soy or corn.
12 . A recombinant CKI polypeptide encoded by the recombinant nucleic acid of claim 1 .
13 . The recombinant nucleic acid of claim 1 , wherein each amino acid substitution is with alanine or serine.