Dna shuffling to produce herbicide selective crops
OwnerVenkiteswaran Subramanian, Willem P.C. Stemmer +3inventors
InventorsVENKITESWARAN SUBRAMANIAN, WILLEM P.C. STEMMER, LINDA A. CASTLE, UMESH S. MUCHHAL, DANIEL L. SIEHL
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Abstract
Methods of shuffling DNA to obtain recombinant herbicide tolerance nucleic acids encoding proteins having new or improved herbicide tolerance activities, libraries of shuffled herbicide tolerance nucleic acids, transgenic plants and DNA shuffling mixtures are provided.
Claims60
A method of obtaining a recombinant herbicide tolerance nucleic acid which can confer tolerance to an herbicide upon a plant in which the recombinant herbicide tolerance nucleic acid is present, the method comprising:
- (i) recombining a plurality of variant forms of one or more parental nucleic acid, wherein the plurality of variant forms comprises segments derived from the parental nucleic acid, wherein the parental nucleic acid encodes an herbicide tolerance activity, or can be shuffled to encode an herbicide tolerance activity, and wherein the plurality of variant forms differ from each other in at least one nucleotide, to produce a library of recombinant nucleic acids;
- (ii) screening the library to identify at least one recombinant herbicide tolerance nucleic acid, wherein the recombinant herbicide tolerance nucleic acid encodes an activity which confers herbicide tolerance to a cell.
A DNA shuffling mixture comprising at least three homologous DNAs, wherein each of the at least three homologous DNAs is derived from a parental nucleic acid encoding a polypeptide or polypeptide fragment selected from the group consisting of:
- a P450 monooxygenase, a glutathione sulfur transferase, a homoglutathione sulfur transferase, a glyphosate oxidase, a phosphinothricin acetyl transferase, a dichlorophenoxyacetate monooxygenase, an acetolactate synthase, a protoporphyrinogen oxidase, a 5-enolpyruvylshikimate-3-phosphate synthase, and a UDP-N-acetylglucosamine enolpyruvyltransferase.
A method of acquiring or improving an herbicide tolerance activity in a parental plant cell, comprising performing whole genome shuffling of a plurality of genomic nucleic acids in the plant cell, forming a modified plant cell, and screening the modified plant cell for a distinct or improved tolerance activity to one or more herbicide compared to the parental plant cell.
A method of predicting long-term efficacy of a herbicide in killing a plant, the method comprising:
- (i.) transforming a plurality of cells of the plant with a library of DNA fragments at least some of which undergo recombination with segments in the genome of the cells to produce modified plant cells;
- (ii.) propagating the modified plant cells in a media containing the herbicide, and recovering surviving plant cells;
- (iii.) recombining DNA from surviving plant cells with a further library of DNA fragments at least some of which undergo recombination with cognate segments in the DNA from the surviving plant cells to produce further modified plant cells;
- (iv.) propagating further modified plant cells, in media containing the herbicide, and collecting further surviving plant cells;
- (v.) repeating (iii.) and (iv.), as necessary, until a further surviving plant cell has acquired a desired degree of resistance to the herbicide, whereby the degree of resistance acquired and the number of repetitions of (iii.) and (iv.) needed to acquire it provide a measure of the long-term efficacy of the herbicide in killing the plant.
Dependencies are inferred from the claim text.