Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide contains a tissue – organ – or cell...
Reexamination Certificate
1998-11-02
2001-08-28
Hutzell, Paula (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide contains a tissue, organ, or cell...
C435S069100, C435S320100, C435S419000, C435S468000, C536S023600, C536S024100, C800S298000
Reexamination Certificate
active
06281409
ABSTRACT:
The present invention relates to transgenic plant production and the expression of gene sequences introduced by genetic transformation procedures. In particular the present invention relates to blackcurrant (
Ribes nigrum
L.) fruit-specific gene promoters and their use in the expression of nucleic acid sequences in transgenic fruit.
Studies on the molecular basis of fruit ripening have concentrated on species whose fruit exhibit a climacteric pattern of ripening, for example tomato, avocado, apple, kiwifruit, peach and mango. Ripening in the fruit from these species is accompanied by a burst in the rate of respiration and a generally large increase in the rate of biosynthesis of the plant growth regulator, ethylene.
Non-climacteric fruit have a considerably different ripening mechanism. Examples of non-climacteric fruit are blueberry, cucumber, grape, orange and strawberry.
Fruit ripening is an important area of scientific research with particular attention being paid to high value fruits such as tomato, kiwifruit and avocado. In the tomato some of the genes involved in the ripening process have been isolated and characterised, for example the gene for polygalacturonase, an enzyme which acts on cell wall pectin. The level of expression of the polygalacturonase gene has been down-regulated in transgenic tomato fruit resulting in increased fruit firmness and consequently extended storage life (Schuch et al, 1991).
In contrast, less is known about the molecular basis of fruit ripening in non-climacteric fruit. In the work leading to the present invention we have found from measurements of respiration rate that blackcurrant fruit do not exhibit a respiratory climacteric during ripening and that ripe fruit produce very low levels of ethylene, hence blackcurrant can be classed as a non-climacteric fruit.
The blackcurrant is the most widely grown bush fruit in Europe, valued particularly for its high content of ascorbic acid and anthocyanin pigments. Areas for potential improvement in blackcurrants include enhancing pigment levels, aroma, flavour, texture, nutritional values (e.g. vitamin content), storage life, weather resistance, pest or pesticide resistance and manipulating sugar, soluble solids or acid levels in the fruit.
Plants with novel/improved characteristics can be produced by introducing genes or DNA sequences from the same or a different organism. Many examples are now in the literature of plant DNA sequences which have been used to drive the expression of foreign genes in plants. In most instances the regions adjacent to the 5′ terminus of the coding regions of genes have been used in gene constructs. These regions are referred to as promoter sequences. In order to produce novel phenotypes it is necessary to have active expression of the introduced DNA sequence by cloning the sequence downstream of a promoter sequence active in plant tissue. These promoters may be derived from plant DNA or from other sources e.g. viruses. In most cases sequences up to 500-1000 bases are sufficient to allow for the regulated expression of foreign genes. However sequences longer than 1 kb may have useful features which permit high levels of gene expression in transgenic plants. Examples of fruit-specific promoters isolated from climacteric fruit such as tomato include the 2A
11
promoter, and the polygalacturonase gene promoter.
Of considerable importance to the development of genetically improved blackcurrants is the finding in the work of the present invention that blackcurrant is in fact a non-climacteric fruit.
Promoters can vary in the level of expression and in the tissue-specific or developmental stage-specific pattern of expression that they drive. Some promoters are expressed in a tissue-specific or developmental stage-specific manner whereas others are expressed in each and every cell and are called constitutive promoters.
The most widely used constitutive promoters are the Cauliflower Mosaic Virus (CaMV) 35S promoter, nopaline synthetase (nos) and the octopine synthetase (ocs) promoters. Due to the different molecular mechanisms of ripening between climacteric and non-climacteric fruit it is hardly appropriate to use fruit-specific promoters isolated from climacteric fruit such as tomato (e.g. the 2A
11
promoter or the polygalacturonase gene) in non-climacteric fruit.
Climacteric fruit-specific promoters therefore may not be suitable for many potential biotechnological applications for the improvement of non-climacteric fruit such as the blackcurrant which ideally require high levels of fruit-specific expression. In the case of the commonly used constitutive promoters, they have the disadvantage that they drive expression at high levels in all or nearly all cell types and throughout the development of the plant. Expression of the introduced gene or DNA sequence driven by a constitutive promoter can have a deleterious effect on normal plant development. Additionally, the commonly used constitutive promoters are derived from plant infectious agents such as plant viruses or Agrobacterium, a soil-borne infectious bacteria. The source of these promoters is a cause for concern in risk assessment of transgenic plant production.
SUMMARY OF THE INVENTION
Accordingly, the present invention provides promoters and a process for obtaining promoters capable of driving fruit-specific expression of DNA sequences in transgenic blackcurrant and other non-climacteric fruit. The process is as defined in claim
1
and the promoters as defined in claim
2
. Preferably the promoter comprises the sequence of nucleic acid bases in
FIG. 9
or IDSEQ 11 herein designated the RIBI promoter or in IDSEQ 14 herein designated the RIB 7 promoter. No previous promoters have been reported to be suitable to drive fruit-specific expression in blackcurrant and other non-climacteric fruit.
One advantage of the present invention is that because of the developmental stage specificity of the expression ie. it offers high level expression in fruit and only very low levels in other tissues, there is a reduced chance that the introduced DNA sequences will have an adverse effect on normal plant development.
The promoters of the present invention also have the advantage over some constitutive promoters in that they are naturally occurring plant gene sequences derived from blackcurrants, ie. a plant that is consumed by humans and not from plant pests or other infectious agents; this overcomes objections to the use of such sequences due to potential recombination.
DETAILED DESCRIPTION OF THE INVENTION
The isolation and characterisation of blackcurrant fruit-specific gene promoters and how they can be used to drive the expression of genes of interest in plants is given below and in the following examples. This description is purely for the purpose of illustrating the invention. It should be noted that the gene promoter may function in a similar (that is, fruit-specific) manner in other related species of non-climacteric fruit, in particular other Ribes species.
Promoters for use in the invention may be isolated from genomic libraries by the use of cDNA probes. The cDNA clones of genes highly expressed specifically in ripe blackcurrant fruit were obtained by differentially screening a cDNA library constructed from mRNA isolated from ripening blackcurrant fruit.
In a further aspect of the invention there is also provided cDNA for genes which exhibit differential expression in fruit during the ripening period of fruit development. In particular the cDNA is identified herein as pRIB1, pRIB3, pRIB5, pRIB6 and pRIB7.
The promoters of the present invention can be used to control the expression of one or more genes in non-climacteric and/or climacteric fruit. Preferably the non-climacteric fruit is the blackcurrant. Suitably the genes are novel/exogenous.
According to the present invention we also provide the use of promoters of the present invention in the transformation of plant cells to control the expression of one or more genes in non-climacteric/climacteric fruit.
In a further aspect of the invention there are provided
Brennan Rex Michael
Taylor Mark Andrew
Woodhead Mary Rose
Hutzell Paula
King William T.
Kinzig Charles M.
Majarian William R.
Mehta Ashwin D.
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