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Root Promoters

Summary

Pioneer Hi-Bred had filed patent applications related to plant promoters containing elements that drive the expression of genes of interest in root cells and tissues.

The claims as filed of the patent applications also describe very broad methods for the isolation and characterization of tissue-specific plant promoters in general. If the claims as filed are granted without change they would potentially cover processes for the identification of tissue-specific promoters regardless of the tissue where the expression of nucleotide sequences is sought.

Pioneer Hi-Bred has now obviously abandoned this patent, although it is still pending in Australia.

The Invention

Pioneer Hi-Bred‘s patent applications in Australia, the U.S. and Europe cover the following aspects:

  • Methods for identifying and isolating tissue-preferred plant promoter elements in general.
  • The elements are not restricted to any particular tissue in the claims as filed.
  • Plant promoters containing root-preferred promoter elements that enhance or suppress the expression of a linked sequence in root cells.
  • Specific sequences of root-promoter elements are spelled out in the claims as filed. Some plant promoters of the invention have multiple root-preferred promoter elements.
  • Methods for root-preferred expression of genes in plants by transforming a plant with an expression cassette having a promoter with elements for root expression; and
  • Plant cells stably transformed with DNA construct containing root-preferred expression elements.

The term “root-preferred” means that the expression driven by a plant promoter of the invention is selectively enhanced or suppressed in roots in comparison to other tissues. Root cells and tissues include any part of the roots, and cover primary, lateral and adventitious roots.

Plants to be transformed with the constructs of the invention are not limited to any in particular in the claims as filed.

Patent No Title, Independent Claims and Summary Applicant
AU 2001/32896

  • Filed – 19 January 2001
  • Granted – under examination
  • Lapsed on 31 Aug 2006
 Title – Novel root-preferred promoter elements and method of use

Claim 1
A plant promoter comprising at least one tissue-preferred plant promoter element, said element identified by:
a) providing a first mixture of oligonucleotides each comprising a 5′ flanking sequence, a central random sequence, and a 3′ flanking sequence;
b) contacting said first mixture with a second mixture comprising nuclear proteins from a preferred plant tissue under binding conditions promoting complex formation between said oligonucleotides and said
proteins;
c) separating said formed complexes electrophoretically;
d) isolating said separated complexes in ranges of electrophoretic mobility;
e) amplifying oligonucleotides of said isolated complexes by polymerase chain reaction utilizing primers to said flanking sequences ;
f) providing said amplified oligonucleotides from step e) as the first mixture for a repetition of step a);
g) performing at least a second cycle of steps b-e with said provided oligonucleotides of step f);
h) assessing for a particular range of electrophoretic mobility and quantity of complex formation in progressive cycles of step g);
i) isolating oligonucleotides of a particular range of electrophoretic mobility wherein said range has increased complex formation in step h);
j) operably linking individual oligonucleotides of step i) to a promoter that drives expression in a plant cell, said promoter operably linked to a coding sequence in an expression cassette;
k) assessing tissue-preferred expression of said coding sequence; and
I) determining sequence of an oligonucleotide having tissue preferred expression in step k).
Claim 5
A plant promoter comprising at least one root-preferred plant promoter element comprising a nucleotide sequence selected from the group consisting of:
a) a nucleotide sequence of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, or SEQ ID NO.8;
b) a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of a); and
c) a nucleotide sequence comprising at least 7 contiguous nucleotides of a sequence of a), wherein said contiguous nucleotides maintain function of the nucleotide sequence of a).
Claim 10
A plant promoter comprising at least one multimeric root-preferred promoter element comprising at least two root-preferred promoter elements further comprising a nucleotide sequence selected from the group consisting of :
a) a nucleotide sequence of SEQ ID NO.1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4,SEQ ID NO.5, SEQ ID NO. 6,SEQ ID NO. 7, or SEQ ID NO. 8;
b) a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of a); and
c) a nucleotide sequence comprising at least 7 contiguous nucleotides of a sequence of a), wherein said contiguous nucleotides maintain function of the nucleotide sequence of a).
Claim 11
A plant promoter comprising at least one root-preferred plant promoter element that enhances expression of a coding sequence operably linked to said promoter, wherein said element comprises a nucleotide sequence selected from the group consisting of :
a) a nucleotide sequence of SEQ ID NO.1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6,SEQ ID NO. 7, or SEQ ID NO. 8;
b) a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of a); and
c) a nucleotide sequence comprising at least 7 contiguous nucleotides of a sequence of a), wherein said contiguous nucleotides maintain function of the nucleotide sequence of a).
Claim 12
A plant promoter comprising at least one root-preferred plant promoter element that suppresses expression of a coding sequence operably linked to said promoter, wherein said element comprises a nucleotide sequence selected from the group consisting of :
a) a nucleotide sequence of SEQ ID NO. 1,SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, or SEQ ID NO. 8;
b) a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of a); and
c) a nucleotide sequence comprising at least 7 contiguous nucleotides of a sequence of a), wherein said contiguous nucleotides maintain function of the nucleotide sequence of a).
Claim 13
A transformed plant, or its parts, having stably incorporated into its genome a DNA construct comprising a plant promoter operably linked to a coding sequence, said plant promoter comprising at least one synthetic root-preferred plant promoter element.
Claim 19
A transformed plant cell, said plant cell having stably incorporated into its genome a DNA construct comprising a plant promoter operably linked to a coding sequence, said plant promoter comprising at least one synthetic root-preferred plant promoter element.
Claim 20
A method for root-preferred expression of a nucleotide coding sequence in a plant, said method comprising transforming a plant cell with a transformation vector comprising an expression cassette, said expression cassette comprising a plant promoter operably linked to said nucleotide coding sequence, said plant promoter comprising at least one synthetic root-preferred plant promoter element.
Claim 22
A method for identifying and isolating tissue-preferred promoter elements, said method comprising the steps of :
a) providing a first mixture of oligonucleotides each comprising a 5′ flanking sequence, a central random sequence, and a 3′ flanking sequence;
b) contacting said first mixture with a second mixture comprising nuclear proteins from a preferred plant tissue under binding conditions promoting complex formation between said oligonucleotides and said proteins;
c) separating said formed complexes electrophoretically;
d) isolating said separated complexes in ranges of electrophoretic mobility;
e) amplifying oligonucleotides of said isolated complexes by polymerase chain reaction utilizing primers to said flanking sequences;
f) providing said amplified oligonucleotides from step e) as the first mixture for a repetition of step a);
g) performing at least a second cycle of steps b-e with said provided oligonucleotides of step f) ;
h) assessing for a particular range of electrophoretic mobility and quantity of complex formation in progressive cycles of step g);
i) isolating by cloning, individual oligonucleotides of a particular range of electrophoretic mobility wherein said range has increased complex formation in step h);
j) simultaneous with step i) or as an individual step, operably linking isolated individual oligonucleotides of step i) to a promoter that drives expression in a plant cell, said promoter operably linked to a coding sequence in an expression cassette;
k) assessing tissue-preferred expression of said coding sequence; and
l) determining sequence of an oligonucleotide having tissue preferred expression in step k).

Pioneer Hi-Bred

Remarks

 Related application in the United States (US 2001/047525 A1) has been expressly abandoned. Applications in Europe (EP 1248850 A2) and Canada (CA 2390819) are also lapsed.