US 12,241,076 B2
Gene responsible for cytoplasmic male sterility
Ian Small, Wattle Grove (AU); and Joanna Melonek, Crawley (AU)
Assigned to VILMORIN & CIE, Paris (FR)
Appl. No. 17/429,176
Filed by VILMORIN & CIE, Paris (FR)
PCT Filed Feb. 6, 2020, PCT No. PCT/EP2020/053036
§ 371(c)(1), (2) Date Aug. 6, 2021,
PCT Pub. No. WO2020/161261, PCT Pub. Date Aug. 13, 2020.
Claims priority of application No. 19305141 (EP), filed on Feb. 6, 2019.
Prior Publication US 2022/0098612 A1, Mar. 31, 2022
Int. Cl. C12N 15/82 (2006.01); C07K 14/415 (2006.01); C12Q 1/6895 (2018.01)
CPC C12N 15/8289 (2013.01) [C07K 14/415 (2013.01); C12Q 1/6895 (2013.01); C12Q 2600/13 (2013.01); C12Q 2600/158 (2013.01)] 3 Claims
 
1. A method for identifying a functional Rf gene encoding a protein able to bind to orf279 RNA, wherein the method comprises the steps of:
a. predicting a target RNA sequence for protein encoded by an Rf gene identified in a wheat plant genome according to the PPR code, comprising:
determining all possible ribonucleotides for each amino acid combination at position 5 and 35 of the PPR motif, and
selecting the target RNA sequence scoring highest for RNA binding to the corresponding PPR,
b. aligning the predicted target RNA sequence with SEQ ID NO: 3,
c. identifying the Rf gene, encoding for a protein, able to bind to a target RNA sequence that shows at least 95% identity to SEQ ID NO: 3,
d. optionally optimizing the sequence of the selected Rf protein by changing amino acids at position 5 and 35 of the PPR motif to improve match according to the PPR code with SEQ ID NO: 3, and
e. validating the binding of said protein to orf279 RNA, comprising:
transforming a wheat plant comprising a sterile cytoplasm expressing orf279, with a vector expressing the identified Rf gene; and
detecting the level of orf279 expression in the transformed plant.