TARGETED MODIFICATION OF MALATE DEHYDROGENASE

• US 20130326725A1
• Filed: 05/02/2013
• Published: 12/05/2013
• Est. Priority Date: 05/02/2012
• Status: Active Grant

• Current Assignee: Corteva Agriscience LLC (Corteva, Inc.)
• Original Assignee: Dow Agrosciences LLC (Corteva, Inc.), Sangamo Biosciences, Inc.
• Inventors: Shukla, Vipula, Urnov, Fyodor, Guschin, Dmitry, Bundock, Paul, Gupta, Manju, Jan De Both, Michiel
• Granted Patent: US 9,523,098 B2
• Time in Patent Office: Days
• US Class Current: 800/278
• CPC Class Codes: A01H 6/825 Solanum lycopersicum [tomato]
• CPC Class Codes: C12N 15/8213 Targeted insertion of genes into the plant genome by homologous recombination
• CPC Class Codes: C12N 15/8241 Phenotypically and genetically modified plants via recombinant DNA technology
• CPC Class Codes: C12N 15/8243 involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
• CPC Class Codes: C12N 15/8261 with agronomic (input) traits, e.g. crop yield
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12Y 101/01037 Malate dehydrogenase (1.1.1.37)
• CPC Class Codes: C12Y 301/00 Hydrolases acting on ester bonds (3.1)
• CPC Class Codes: Y02A 40/146 Genetically Modified [GMO] plants, e.g. transgenic plants

Abstract

Disclosed herein are methods and compositions for targeted modification of one or more endogenous plant malate dehydrogenase genes.

Claims

1. A plant comprising one or more plant cells in which an endogenous malate dehydrogenase (MDH) gene is modified such that activity of an expressed MDH protein is modified and wherein crop yield from the plant is increased.

2. The plant of claim 1, wherein the endogenous MDH gene is a mitochondrial MDH gene (mMDH).

3. The plant of claim 1, wherein the modification comprises a mutation in one or more NADH binding domains of the MDH gene.

4. The plant of claim 1, wherein the mutation is within amino acid residues 104-136 and/or 171-220 of SEQ ID NO:

1, or in a comparably numbered and aligned region in a different plant species.

5. The plant of claim 1, wherein one or more alleles of MDH are modified.

6. A plant part from the plant of claim 1, comprising the one or more modified plant cells.

7. The plant part of claim 6, wherein the plant part is a leaf, stem, root, flower, seed, or fruit.

8. The plant of claim 1, wherein the plant is selected from the group consisting of tomato, soy, maize, potato and alfalfa.

9. The plant of claim 1, wherein the modification is made using a fusion protein comprising a DNA-binding domain and a functional domain.

10. The plant of claim 9, wherein the fusion protein is a zinc finger nuclease.

11. The plant of claim 10, wherein the zinc finger nuclease comprises five or six zinc finger domains ordered finger 1 to finger 5 or finger 1 to finger 6, each zinc finger domain comprising a recognition helix region, wherein the zinc finger nuclease comprises the recognition helix regions ordered and shown in a single row of Table 1A.

12. The plant of claim 10, wherein the zinc finger nuclease comprises a non-canonical zinc finger domain.

13. The plant of claim 9, wherein the fusion protein is introduced into the plant cell as a polynucleotide.

14. A method for producing a plant according to claim 1, the method comprising introducing one or more zinc finger nucleases into a plant cell such that the MDH gene is modified in the plant cell.

15. The method of claim 14, wherein the method comprises expressing a pair of fusion proteins each comprising a zinc finger DNA binding domain and a nuclease domain in the plant cell, wherein the pair of fusion proteins dimerize and cleave MDH such that MDH protein activity is reduced.

16. The method of claim 15, further comprising introducing an exogenous nucleic acid sequence into the cell, wherein the exogenous sequence is integrated into the MDH gene.

17. The method of claim 14, wherein the modification comprises a mutation in one or more NADH binding domains.

18. The method of claim 14, wherein the altered MDH protein activity results in increased photosynthesis, modifications to the citric acid cycle within the plant cell, higher levels of malate in the plant cell, reduced OAA levels in the cell and/or increased fruit yield.

19. A method of increasing crop yield, comprising planting a plant of claim 1, or a seed or plant part of the plant.

20. The method of claim 19, wherein the plant is selected from the group consisting of tomato, soy, maize, potato and alfalfa.