Cold-inducible and tuber-specific promoter sequence from...

Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part

Reexamination Certificate

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C435S320100, C435S419000, C435S468000, C536S024100, C800S287000, C800S298000

Reexamination Certificate

active

06184443

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a promoter, including a construct and an expression vector comprising the same and a transformed cell comprising the same. In addition the present invention relates to a plant comprising the same.
BACKGROUND OF THE INVENTION
It is known that it is desirable to direct expression of a gene of interest (“GOI”) in certain tissues of an organism—such as a plant. For example, it may be desirable to produce crop protein products with an optimised amino acid composition and so increase the nutritive value of the crop. It may even be desirable to use the crop to express non-plant genes such as genes for mammalian products. Examples of the latter products include interferons, insulin, blood factors and plasminogen activators.
However, whilst it may be desirable to achieve expression of a GOI in certain tissues it is sometimes important (if not necessary) to ensure that the GOI is not expressed in other tissues in such a manner that detrimental effects may occur. Moreover, it is important not to upset the normal metabolism of the organism to such an extent that detrimental effects occur. For example, a disturbance in the normal metabolism in a plant's leaf or root tip could lead to stunted growth of the plant.
CA-A-2006454 describes a DNA sequence of an expression cassette in which the potato tuber specific regulatory regions are localised. The expression cassette contains a patatin-gene with a patatin-gene promoter. The DNA sequence is transferred into a plant genome using agrobacteria. According to CA-A-2006454, the DNA sequence enables heterologous products to be prepared in crops.
One of the key plant enzymes is &agr;-amylase. &agr;-amylase participates in the pathway responsible for the breakdown of starch to reducing sugars in potato tubers. Genes coding for &agr;-amylase in potato plants have been isolated and characterised. For example, see the teachings in EP-B-0470145.
In brief, &agr;-amylase is encoded by a gene family consisting of at least 5 individual genes. Based on their homology the genes can be divided into two subfamilies—one of which is the type 3 amylase(s), the other of which is the type 1 amylase(s). The two groups of &agr;-amylases are expressed differently, not only on the molecular level but also in different tissues of the potato plant.
In this regard, type 3 &agr;-amylases are expressed in root, in tubers, in sprouts and in stem tissue; whereas type 1 &agr;-amylases are expressed in sprout and stem tissues, but not in tubers.
SUMMARY OF THE INVENTION
The present invention seeks to provide a plant promoter that is capable of directing the expression of a gene of interest in specific tissues, or in just a specific tissue, of an organism, typically a plant.
According to a first aspect of the present invention there is provided a promoter comprising a nucleotide sequence corresponding to the 5.5 Kb EcoR1 fragment isolated from
Solanum tuberosum
, or a variant, homologue or fragment thereof.
A restriction map of the 5.5 Kb EcoR1 fragment isolated from
Solanum tuberosum
is shown in
FIGS. 1
,
2
and
8
—which are discussed later.
According to a second aspect of the present invention there is provided a promoter comprising a nucleotide sequence corresponding to the 5.5 Kb EcoR1 fragment isolated from
Solanum tuberosum
, or a variant, homologue or fragment thereof but wherein at least a part of the promoter is inactivated.
According to a third aspect of the present invention there is provided a promoter comprising at least the nucleotide sequence shown as SEQ ID NO:1or a variant, homologue or fragment thereof.
According to a fourth aspect of the present invention there is provided a promoter comprising the nucleotide sequence of any of one of the sequences shown as Seq.I.D.No.s 4-17, preferably any of one of the sequences shown as Seq.I.D.No.s 4-16, or a variant, homologue or fragment thereof.
According to a fifth aspect of the present invention there is provided a promoter comprising a nucleotide sequence corresponding to the 5.5. Kb EcoR1 fragment isolated from
Solanum tuberosum
, or a variant, homologue or fragment thereof, but wherein at least the nucleotide sequence shown as SEQ ID NO:1 is inactivated.
According to a sixth aspect of the present invention there is provided a promoter comprising a nucleotide sequence corresponding to the 5.5. Kb EcoR1 fragment isolated from
Solanum tuberosum
, or a variant, homologue or fragment thereof, but wherein at least any of one of the sequences shown as SEQ ID NOS:2-16 is inactivated.
According to a seventh aspect of the present invention there is provided a construct comprising the promoter according to the present invention fused to a GOI.
According to an eighth aspect of the present invention there is provided an expression vector comprising the promoter according to the present invention.
According to a ninth aspect of the present invention there is provided a transformation vector comprising the promoter according to the present invention.
According to a tenth aspect of the present invention there is provided a transformed cell comprising the promoter according to the present invention.
According to an eleventh aspect of the present invention there is provided a transgenic organism comprising the promoter according to the present invention.
According to a twelfth aspect of the present invention there is provided the use of the promoter according to the present invention as a cold inducible promoter.
According to a thirteenth aspect of the present invention there is provided a construct comprising the promoter of the present invention and a nucleotide sequence coding for anti-sense alph&agr;-amylase.
According to a fourteenth aspect of the present invention there is provided the use of a promoter according to the present invention for expressing a GOI in tuber and/or sprout and/or root and/or stem of a plant, preferably in just or at least tuber of a plant.
Other aspects of the present invention include methods of expressing or transforming any one of the expression vector, the transformation vector, the transformed cell, including in situ expression within the transgenic organism, as well as the products thereof. Additional aspects of the present invention include uses of the promoters for expressing GOIs in vitro (e.g. in culture media such as a broth) and in vivo (e.g. in a transgenic organism).
Preferably, in any one of the expression vector, the transformation vector, the transformed cell or the transgenic organism the promoter is present in combination with at least one GOI.
Preferably the transformation vector is derived from agrobacterium.
Preferably the promoter is stably incorporated within the transgenic organism's genome.
Preferably the transgenic organism is a plant. Preferably the plant is a dicot plant.
More preferably, the plant is a potato plant.


REFERENCES:
patent: 5436393 (1995-07-01), Rocha-Sosa et al.
patent: 5705375 (1998-01-01), Van Ooyen et al.
patent: 2006454 (1989-12-01), None
patent: 120 516 A2 (1984-02-01), None
patent: 470 145 B1 (1990-04-01), None
patent: 455 316 A2 (1991-04-01), None
patent: WO 90/12876 (1990-11-01), None
patent: WO 92/05259 (1992-04-01), None
Yamaguchi-Shinozaki K, et al. “A novel cis-acting element in an Arabidopsis gene is involved in responsiveness to drought, low-temperature, or high-salt stress.” Plant Cell 6: 251-264, Feb. 1994.
Baker SS, et al. “The 5′region ofArabidopsis thalianacor15a has cis-acting elements that confer cold-, drought- and ABA-regulated gene expression.” Plant Mol. Biol. 24: 701-713, Mar. 1994.
Dolferus R, et al. “Regulation of the Arabidopsis Adh gene by anaerobic and other environmental stresses.” Ann. Bot. 74: 301-308, Sep. 1994.
Benfey PN, et al. “The cauliflower mosaic virus 35S promoter: Combinatorial regulation of transcription in plants.” Science 250: 959-966, Nov. 1990.
Kim Y, et al. “A 20 nucleotide upstream element is essential for the nopaline synthase (nos) promoter activity.” Plant Mol. Biol. 24: 105-117, 1994.
Fraley, Robert T.,et al.; CRC Critical Re

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