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
1997-08-14
2001-03-20
Smith, Lynette R. F. (Department: 1649)
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
C536S024100, C800S298000, C800S317000, C800S278000, C800S283000, C800S287000, C435S173300, C435S320100
Reexamination Certificate
active
06204437
ABSTRACT:
The present invention relates to DNA constructs and plants incorporating them. In particular, it relates to promoter sequences for the expression of genes in plants.
Gene expression is controlled by various regulatory components, including nucleic acid and protein elements. In particular, gene expression is controlled by a region commonly referred to as the “promoter” which lies upstream (5′) of the protein encoding region. A promoter may be constitutive or tissue-specific, developmentally-regulated and/or inducible.
Within the promoter region there are several domains which are necessary for full function of the promoter. The first of these domains lies immediately upstream of the structural gene and forms the “core promoter region” containing consensus sequences, normally 70 base pairs immediately upstream of the gene. The core promoter region contains the characteristic CAAT and TATA boxes plus surrounding sequences, and represents a transcription initiation sequence which defines the transcription start point for the structural gene. The precise length of the core promoter region is indefinite but it is usually well-recognisable. Such a region is normally present, with some variation, in all promoters. The base sequences lying between the various well-characterised “boxes” appear to be of lesser importance.
The presence of the core promoter region defines a sequence as being a promoter: if the region is absent, the promoter is non-functional. Furthermore, the core promoter region is insufficient to provide full promoter activity. A series of regulatory sequences upstream of the core constitute the remainder of the promoter. The regulatory sequences determine expression level, the spatial and temporal pattern of expression and, for an important subset of promoters, expression under inductive conditions (regulation by external factors such as light, temperature, chemicals, hormones).
Manipulation of crop plants to alter and/or improve phenotypic characteristics (such as productivity or quality) requires the expression of heterologous genes in plant tissues. Such genetic manipulation therefore relies on the availability of means to drive and to control gene expression as required; for example, on the availability and use of suitable promoters which are effective in plants and which regulate gene expression so as to give the desired effect(s) in the transgenic plant. It is advantageous to have the choice of a variety of different promoters so that the most suitable promoter may be selected for a particular gene, construct, cell, tissue, plant or environment.
Promoters (and other regulatory components) from bacteria, viruses, fungi and plants have been used to control gene expression in plant cells. Numerous plant transformation experiments using DNA constructs comprising various promoter sequences fused to various foreign genes (for example, bacterial marker genes) have led to the identification of useful promoter sequences. It has been demonstrated that sequences up to 500-1000 bases in most instances are sufficient to allow for the regulated expression of foreign genes. However, it has also been shown that sequences much longer than 1 kb may have useful features which permit high levels of gene expression in transgenic plants. A range of naturally-occurring promoters are known to be operative in plants and have been used to drive the expression of heterologous (both foreign and endogenous) genes in plants: for example, the constitutive 35S cauliflower mosaic virus promoter, the ripening-enhanced tomato polygalacturonase promoter (Bird et al, 1988, Plant Molecular Biology, 11:651-662), the E8 promoter (Diekman & Fischer, 1988, EMBO, 7:3315-3320) and the fruit specific 2A11 promoter (Pear et al, 1989, Plant Molecular Biology, 13:639-651) and many others.
As stated above, successful genetic manipulation relies on the availability of means to control plant gene expression as required. The scientist uses a suitable expression cassette (incorporating one or more promoters and other components) to regulate gene expression in the desired manner (for example, by enhancing or reducing expression in certain tissues or at certain developmental stages). The ability to choose a suitable promoter from a range of promoters having differing activity profiles is thus important.
One object of the present invention is to provide alternative promoters capable of driving gene expression in plants. Such promoters are suitable for incorporation into DNA constructs encoding any target gene so that the target gene is expressed when the construct is transformed into a plant. It may be particularly advantageous to provide alternative promoters which exhibit particular spatial or temporal patterns of expression, for example promoters which are active in certain cell-types and/or are particularly responsive to certain developmental events and environmental conditions. This may allow more selective control of gene expression and its effects, as the target gene is only activated where and/or when it is required.
In work leading to the present invention, we have isolated and fully sequenced three ACC oxidase gene promoters from tomato. ACC oxidase is an enzyme involved in the biosynthesis of ethylene.
Ethylene is a major plant hormone which has been shown to have a variety of effects on plant growth and development in many species. Endogenous levels of ethylene increase during several stages of development and in response to various stimuli including mechanical wounding and pathogen infection, ripening of climacteric fruits and leaf and flower senescence. The biosynthetic pathway for ethylene in plants is well-established; for example, a review of ethylene biosynthesis was published by Yang and Hoffman in 1984 (Annual Review Plant Physiology, 35:155-189). The final stages of ethylene biosynthesis proceed by the following pathway:
Methionine→
S-adenosyl-L-methionine (SAM)→
1-aminocyclopropane-1-carboxylic acid (ACC)→Ethylene.
The final step in the pathway of ethylene biosynthesis is the conversion of the cyclic amino acid 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene. This reaction is catalysed by the enzyme ACC oxidase (also know as ethylene forming enzyme or EFE) which was once thought to be constitutively expressed in most tissues. However, since the cloning of the gene the messenger RNA has been shown to be induced under a number of conditions known to result in increased ethylene production (Davies and Grierson, 1989, Planta, 179:73-80; Hamilton et al, 1990, Nature, 346:284-287).
In tomato, ACC oxidase is encoded by a multigene family comprising three members, hereinafter called the Aco1 gene, the Aco2 gene and the Aco3 gene. Bouzayen et al (1993, pp 76-81 in Cellular and molecular aspects of the plant hormone ethylene, eds. Pech et al, Kluwer Academic Publishers, NL) discuss the expression and characterisation of the ACC oxidase (EFE) multigene family in tomato plants, and
FIG. 1
shows the structure and similarity of the gene family. When the open reading frame regions of the three tomato ACC oxidase (Aco) genes are aligned, the overall identity is 79.3%. The 5′ or 3′ un-translated regions are less homologous than the coding regions.
The coding region of the Aco1 tomato gene corresponds to the TOM13 cDNA clone, first described as a ripening-related clone by Slater et al (1985, Plant Molecular Biology, 5:137-147).
The coding region of the Aco2 tomato gene corresponds to the gTOMA gene sequence published by Holdsworth et al in Nucleic Acids Research, 1987, 15:10600.
The Aco3 tomato gene is equivalent to the clone gTOMB, described (without sequence data) in Holdsworth et al, 1988, Plant Molecular Biology, 11:81-88. Because no expression of gTOMB (Aco3) was detected, Holdsworth et al suggested this was a pseudogene, thus suggesting that an active and useful promoter could not be isolated from this gene.
After the cloning of the first ACC oxidase cDNA clone (pTOM13), standard hybridisation procedures were used to isolate clones for ACC oxidase from other plant s
Barry Cornelius
Blume Beatrix
Grierson Donald
Hamilton Andrew
Holdsworth Michael
Hohenschutz Liza D.
Kimball Melissa L.
Smith Lynette R. F.
Zeneca Limited
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