Methods and compositions for the introduction and regulated expression of genes in plants

Patent metadata

Field	Value
Publication number	US-9765352-B2
Application number	US-201314087775-A
Country	US
Kind code	B2
Filing date	Nov 22, 2013
Priority date	Dec 30, 2009
Publication date	Sep 19, 2017
Grant date	Sep 19, 2017

Abstract

Compositions and methods are provided for the introduction and the regulated expression of genes in plants. Compositions include promoter constructs that provide a level of activity useful for the regulated expression of site-specific recombinases, while avoiding premature excision. Further provided are isolated polynucleotides encoding novel babyboom polypeptides, expression cassettes, and plants comprising the same. Methods for the introduction of genes into plants are provided, including methods for plastid transformation and methods for the transformation of tissues from mature seeds and leaves.

First claim

That which is claimed:

1. A promoter construct comprising a promoter followed by a first attachment B (attB) site, wherein the promoter is selected from the group consisting of:
a) a promoter comprising a nucleotide sequence having the sequence set forth in SEQ ID NO: 29; and
b) a promoter comprising a nucleotide sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO: 29;
wherein said first attB site has the nucleotide sequence set forth in SEQ ID NO: 31 and further wherein said first attB site decreases the activity of said promoter.

2. The promoter construct of claim 1 , wherein said promoter comprises the sequence set forth in nucleotides 291-430 of SEQ ID NO: 29 or a sequence having at least 85% sequence identity to the sequence set forth in nucleotides 291-430 of SEQ ID NO: 29.

3. The promoter construct of claim 1 , wherein said promoter construct further comprises a linker sequence that separates said promoter and said first attB site.

4. The promoter construct of claim 3 , wherein said linker sequence that separates said promoter and said first attB site is about 133 nucleotides in length.

5. The promoter construct of claim 3 , wherein said linker sequence that separates said promoter and said first attB site comprises nucleotides of a maize rab17 5′ untranslated region (5′-UTR).

6. The promoter construct of claim 5 , wherein said linker sequence that separates said promoter and said first attB site comprises the nucleotide sequence set forth in SEQ ID NO: 35.

7. The promoter construct of claim 3 , wherein said linker sequence that separates said promoter and said first attB site has the sequence set forth in SEQ ID NO: 36 or a nucleotide sequence having at least 70% sequence identity to the sequence set forth in SEQ ID NO: 36.

8. An expression cassette comprising the promoter construct of claim 1 operably linked to a polynucleotide of interest.

9. The expression cassette of claim 8 , wherein said polynucleotide of interest is a nucleotide sequence encoding a site-specific recombinase.

10. The expression cassette of claim 9 , wherein said site-specific recombinase is selected from the group consisting of FLP, Cre, SSV1, lambda Int, phi C31 Int, HK022, R, Gin, Tn1721, CinH, ParA, Tn5053, Bxb1, TP907-1, and U153.

11. The expression cassette of claim 8 , further comprising a polynucleotide encoding a babyboom polypeptide operably linked to a second promoter, wherein said second promoter is active in a plant.

12. The expression cassette of claim 11 , wherein said babyboom polypeptide comprises at least two AP2 domains and at least one of the following amino acid sequences:
a) the amino acid sequence set forth in SEQ ID NO: 54 or an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 54 by one amino acid; and
b) the amino acid sequence set forth in SEQ ID NO: 57 or an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 57 by one amino acid.

13. The expression cassette of claim 11 , wherein said polynucleotide encoding said babyboom polypeptide has a nucleotide sequence selected from the group consisting of:
a) the nucleotide sequence set forth in SEQ ID NO: 13;
b) a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 13;
c) a nucleotide sequence encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO: 14; and
d) a nucleotide sequence encoding a polypeptide having an amino acid sequence having at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14.

14. The expression cassette of claim 11 , wherein said second promoter is a maize ubiquitin promoter or a maize oleosin promoter.

15. The expression cassette of claim 11 , wherein said expression cassette further comprises a polynucleotide encoding a Wuschel polypeptide operably linked to a third promoter, wherein said third promoter is active in a plant.

16. The expression cassette of claim 15 , wherein said polynucleotide encoding said Wuschel polypeptide has a nucleotide sequence selected from the group consisting of:
a) the nucleotide sequence set forth in SEQ ID NO: 63; and
b) a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 63;
c) a nucleotide sequence encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO: 64; and
d) a nucleotide sequence encoding a polypeptide having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 64.

17. The expression cassette of claim 15 , wherein said third promoter is a maize In2-2 promoter or a nopaline synthase promoter.

18. A host cell comprising the expression cassette of claim 8 .

19. A plant cell comprising the expression cassette of claim 8 .

20. A transgenic seed of a plant comprising the plant cell of claim 19 , wherein said seed comprises said expression cassette.

21. A plant cell comprising an expression cassette comprising a promoter construct according to claim 1 operably linked to a polynucleotide encoding a site-specific recombinase, wherein said plant cell further comprises a polynucleotide of interest flanked by a first and a second recombination site, wherein said first and second recombination sites are recombinogenic with respect to one another and are directly repeated, and wherein said site-specific recombinase can recognize and implement recombination at said first and said second recombination sites, thereby excising said polynucleotide of interest.

22. A method for expressing a polynucleotide of interest in a plant cell, said method comprising introducing into said plant cell an expression cassette, wherein said expression cassette is the expression cassette according to claim 8 .

23. A method for excising a polynucleotide of interest from a target site in a plant cell, wherein said target site comprises a first site-specific recombination site, said polynucleotide of interest, and a second site-specific recombination site, wherein said first and said second site-specific recombination sites are recombinogenic with respect to one another and are directly repeated, said method comprising:
a) introducing into said plant cell an expression cassette comprising a first promoter operably linked to a polynucleotide encoding a site-specific recombinase, wherein said first promoter comprises the promoter construct of claim 1 ; and
b) expressing said polynucleotide encoding said site-specific recombinase, wherein said site-specific recombinase recognizes and implements recombination at said first and said second site-specific recombination sites, thereby excising said polynucleotide of interest.

24. The method of claim 23 , wherein said target site comprises in operable linkage: said first recombination site, a second promoter, a polynucleotide sequence encoding a Wuschel polypeptide, a third promoter, a polynucleotide encoding a babyboom polypeptide, and said second recombination site, wherein said polynucleotide encoding said Wuschel polypeptide and its operably linked second promoter can follow or precede said polynucleotide encoding said babyboom polypeptide and its operably linked third promoter in the target site.

25. A method for excising a polynucleotide of interest from a target site in a plant cell, wherein said target site comprises in operable linkage: a first site-specific recombination site, a first promoter, said polynucleotide of interest, a second promoter, a

Assignees

• Pioneer Hi Bred Int
• Du Pont
• Du Pont
• Pioneer Hi Bred Int

Inventors

• Gordon-Kamm William James
• Klein Theodore Mitchell
• Lowe Keith S
• Mcbride Kevin E
• Scelonge Christopher Jay
• Wang Ning

Classifications

• C12N15/8241 Primary

  Phenotypically and genetically modified plants via recombinant DNA technology · CPC title

• C12N15/8262

  involving plant development · CPC title

• C12N15/8216

  Methods for controlling, regulating or enhancing expression of transgenes in plant cells · CPC title

• C12N15/8213 Primary

  Targeted insertion of genes into the plant genome by homologous recombination · CPC title