Modulation of Transgene Expression in Plants

Patent metadata

Field	Value
Publication number	US-2018100162-A1
Application number	US-201715705312-A
Country	US
Kind code	A1
Filing date	Sep 15, 2017
Priority date	Oct 11, 2016
Publication date	Apr 12, 2018
Grant date	—

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 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 20 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 miR1512 or miR3522.

10 . The transgenic plant tissue, plant part, or plant of claim 9 , wherein the target site is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, and combinations thereof.

11 . 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.

12 . 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.

13 . 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 for at least one sRNA molecule; and transforming a plant cell with the expression cassette, wherein the plant cell expresses the sRNA.

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

15 . The method of claim 13 , 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.

16 . The method of claim 13 , wherein the sRNA is miR1512 or miR3522.

17 . The method of claim 16 , wherein the target site is a nucleic acid having at least 90% sequence identity to a nucleic acid selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, and combinations thereof.

18 . The method of claim 13 , wherein the sRNA is encoded by a polynucleotide that is operably linked to a non-constitutive promoter in the genome of the plant cell.

19 . The method according to claim 18 , wherein the non-constitutive promoter is a tissue-preferred, tissue-specific, or cell type-specific.

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

21 . The method according to claim 20 , wherein the tissue-specific promoter initiates transcription selectively in vegetative tissue.

22 . A plant tissue comprising a plant cell produced by the method according to claim 13 .

23 . A plant comprising the plant tissue of claim 22 .

24 . 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; providing a plant cell, wherein the sRNA is expressed predominantly in seed tissue; and transforming the plant cell with the expression cassette.

25 . The method of claim 24 , wherein the sRNA is miR1512.

26 . The method of claim 25 , wherein the target site is SEQ ID NO:4.

27 . 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 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.

28 . The method of claim 27 , 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.

29 . The method of claim 27 , wherein the plant is a dicot plant selected from the group consisting of soybean, canola, cotton, and tobacco.

30 . The method of claim 27 , 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.

31 . The method of claim 30 , wherein the endogenous sRNA is expressed predominantly in seed tissue.

32 . The method of claim 31 , wherein the sRNA is miR1512.

33 . The method of claim 32 , wherein the target site is SEQ ID NO:4.

Assignees

• Dow Agrosciences Llc

Inventors

• Kumar Sandeep
• German Marcelo Ariel
• Wang Pohao
• 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

• C12N15/8218 Primary

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

• C12N15/8274

  for herbicide resistance · CPC title

• C12N15/8223

  Vegetative tissue-specific promoters · CPC title

• Y02A40/146

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