Modulation of transgene expression in plants

Patent metadata

Field	Value
Publication number	US-11473095-B2
Application number	US-202016856536-A
Country	US
Kind code	B2
Filing date	Apr 23, 2020
Priority date	Oct 11, 2016
Publication date	Oct 18, 2022
Grant date	Oct 18, 2022

Abstract

This disclosure concerns the use of endogenous plant RNAi machinery to preferentially or specifically reduce transgene expression. In some embodiments, the disclosure concerns specific reduction of transgene expression in seed tissues of a dicot plant.

First claim

What is claimed is:

1. A nucleic acid expression cassette comprising a gene of interest, a plant promoter operably linked to the gene of interest, and a target site, wherein the target site comprises SEQ ID NO:4, for at least one sRNA molecule present in a dicot plant.

2. The expression cassette of claim 1 , wherein the gene of interest is an agronomic trait gene, an insect resistance gene, an herbicide tolerance gene, a non-coding RNA gene, or a selectable marker gene.

3. The expression cassette of claim 1 , wherein the target site is between about 21 and about 25 nucleotides in length.

4. The expression cassette of claim 1 , wherein the dicot plant is selected from the group consisting of Arabidopsis , soybean, rapeseed, cotton, and tobacco.

5. A transgenic plant cell comprising the expression cassette of claim 1 , wherein the gene of interest is heterologous to the plant cell, and wherein the at least one sRNA molecule is an endogenous sRNA of the plant cell.

6. The transgenic plant cell of claim 5 , wherein the endogenous sRNA is expressed in the plant in a tissue-specific, tissue-preferred, or cell type-specific manner.

7. A transgenic plant tissue, plant part, or plant, comprising the transgenic plant cell of claim 5 .

8. The transgenic plant tissue, plant part, or plant of claim 7 , wherein the endogenous sRNA is expressed predominantly in seed tissue.

9. The transgenic plant tissue, plant part, or plant of claim 8 , wherein the sRNA is SEQ ID NO:3.

10. The transgenic plant tissue, plant part, or plant of claim 9 , wherein the gene of interest is an herbicide tolerance gene, and wherein expression of the herbicide tolerance gene is downregulated in seed tissue.

11. The transgenic plant tissue, plant part, or plant of claim 7 , wherein the target site is modified for partial or complete downregulation of the gene of interest.

12. A method for producing a transgenic plant cell, the method comprising:
providing a nucleic acid expression cassette comprising an herbicide tolerance gene, a plant promoter operably linked to the gene, and a target site, wherein the target site comprises SEQ ID NO:4, for at least one sRNA molecule; and
transforming a plant cell with the expression cassette, wherein the plant cell expresses the sRNA.

13. The method of claim 12 , wherein the sRNA hybridizes to mRNA transcribed from the herbicide tolerance gene and thereby reduces the expression of the herbicide tolerance gene.

14. The method of claim 12 , wherein the sRNA is an endogenous sRNA that is expressed in the plant cell in a tissue-preferred, tissue-specific, or cell type-specific manner.

15. The method of claim 12 , wherein the sRNA is SEQ ID NO:3.

16. The method of claim 12 , wherein the sRNA is encoded by a polynucleotide that is operably linked to a non-constitutive promoter in the genome of the plant cell.

17. The method according to claim 16 , wherein the non-constitutive promoter is a tissue-preferred, tissue-specific, or cell type-specific.

18. The method according to claim 16 , wherein the non-constitutive promoter is a tissue-specific promoter.

19. The method according to claim 18 , wherein the tissue-specific promoter initiates transcription selectively in vegetative tissue.

20. A plant tissue comprising a plant cell produced by the method according to claim 12 .

21. A plant comprising the plant tissue of claim 20 .

22. A method for producing a transgenic plant cell, the method comprising:
providing a nucleic acid expression cassette comprising a gene of interest and a target site of a sRNA molecule, wherein the target site comprises SEQ ID NO:4;
providing a plant cell, wherein the sRNA is expressed predominantly in seed tissue; and
transforming the plant cell with the expression cassette.

23. The method of claim 22 , wherein the sRNA is SEQ ID NO:3.

24. A method for inhibiting expression of a gene of interest in a plant, comprising growing a plant comprising a nucleic acid expression cassette comprising a gene of interest, a plant promoter operably linked to the gene of interest, and a target site, wherein the target site comprises SEQ ID NO:4, for at least one sRNA molecule present in the plant, wherein expression of the gene of interest is downregulated only in tissues where the sRNA molecule is expressed.

25. The method of claim 24 , wherein the gene of interest is an agronomic trait gene, an insect resistance gene, an herbicide tolerance gene, a non-coding RNA gene, or a selectable marker gene.

26. The method of claim 24 , wherein the plant is a dicot plant selected from the group consisting of soybean, canola, cotton, and tobacco.

27. The method of claim 24 , wherein the gene of interest is heterologous to the plant, and wherein the at least one sRNA molecule is an endogenous sRNA of the plant.

28. The method of claim 27 , wherein the endogenous sRNA is expressed predominantly in seed tissue.

29. The method of claim 28 , wherein the sRNA is SEQ ID NO:3.

Assignees

• Corteva Agriscience Llc

Inventors

• Kumar Sandeep
• German Marcelo Ariel
• Wang Po-Hao
• Glancy Todd P
• Sriram Shreedharan
• Yerkes Carla N
• Bowling Andrew J
• Pence Heather
• Robinson Andrew E

Classifications

• C12N15/8234

  Seed-specific, e.g. embryo, endosperm · CPC title

• C12N15/8274

  for herbicide resistance · CPC title

• C12N15/8218 Primary

  Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS] · CPC title

• C12N15/8223

  Vegetative tissue-specific promoters · CPC title

• Y02A40/146

  Genetically Modified [GMO] plants, e.g. transgenic plants · CPC title