Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1993-12-07
2001-08-07
Nelson, Amy J. (Department: 1638)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
Reexamination Certificate
active
06271033
ABSTRACT:
This invention relates to novel DNA constructs, plant cells containing them and plants derived therefrom. In particular it involves the use of recombinant DNA technology to control, and more specifically to reduce or inhibit, gene expression in plants.
Plant development is a complex physiological and biochemical process requiring the co-ordinated expression of many genes. It produces plants whose products, such as such as roots, foliage, fruit and seeds, are used in agriculture and food production. It has long been the aim of scientists to develop methods which allow the manipulation of these genes for the purposes of producing improved plant varieties. Such varieties may be resistant to insects or herbicides, have improved agronomic performance or be of better quality. For this purpose, many methods have been developed for the isolation of plant genes, and their characterisation both in vitro and in vivo. The in vivo work has been supplemented by the development of transformation techniques. These permit the design of novel plant varieties, having altered and improved phenotypes desired in the agricultural industry.
One objective which has been desirable for many years, but which has only recently been achieved is the inhibition of specific genes which may have deleterious effects on plant growth or performance or the quality of plant products. This has been done by the expression of antisense RNA to the gene which is to be inhibited. A number of examples are now found in the literature which demonstrate that this method can be made to work very effectively in plants. A typical example is disclosed in our European patent specification EP 271988 (equivalent to U.S. Ser. No. 07/119614). This shows the inhibition by antisense RNA of enzymes, in particular polygalacturonase and pectin methylesterase, involved in cell wall modification during tomato fruit ripening.
Antisense RNA is a technique which will now find wide application in the modification of crop plants. Its mechanism is not clearly understood. One theory is that antisense RNA produced in the nucleus of transformed plants will form RNA-RNA hybrid molecules which will lead to the inhibition of the expression of the specific gene to which antisense RNA has been expressed. An alternative plausible hypothesis is that the effects is due to hybrids of RNA with DNA. In experiments carried out so far, enzyme inhibition of greater than 95% has been observed in the primary transformed plants. This level of inhibition has been increased further by genetic experiments, in which the copy number of the antisense gene has been doubled. Thus inhibition of nearly 100% of PG gene expression has been achieved.
Although this method works well, there is the need for the development of other methods which lead to the inhibition of gene expression through alternative molecular pathways in order to widen the repertoire of technical possibilities which will permit the fine-tuning of gene expression in transgenic plants. The present invention provides such an alternative method.
According to the present invention we provide a process for the inhibition of the production of a target gene product in a plant cell which comprises generating in the cell while the target gene is being expressed mRNA from recombinant DNA coding for part only of the gene product. We further provide novel constructs for use in the process of the invention which comprise recombinant DNA coding for part only of a target gene naturally expressed in a cell which DNA is expressed under the control of a promoter sequence operative in plant cells. The invention further comprises novel cells and plants in which the process is realised or which (or ancestors of which) have been transformed with the constructs of the invention.
Inhibition of gene products according to the invention may be partial or almost complete. It is not as yet clear why the invention works. The almost complete inhibition of gene expression that can be obtained under certain circumstances is particularly surprising.
DNA constructs according to the invention preferably comprise a DNA coding base sequence at least 50 bases in length. The upper limit to the base sequence depends on the size of the gene whose product is to be inhibited. The theoretical upper limit will generally have to be established by trial and error in each case. However for most practical purposes it is unnecessary to establish this upper limit, as for convenience it will generally be found suitable to use sequences between 100 and 1000 bases in length. The preparation of such constructs is described in more detail below. Many constructs suitable for use in the process of the present invention are described in U.S. Ser. No. 07/119,614 (the entire disclosure of which specification is incorporated herein by reference). In that specification they are referred to as “sense” constructs and generally given the suffix “S”.
According to the invention we propose to use both constitutive promoters (such as cauliflower mosaic virus 35S RNA) and inducible or developmentally regulated promoters (such as the ripe-fruit-specific polygalacturonase promoter) as circumstances require. Use of a constitutive promoter will tend to affect functions in all parts of the plant in which the target gene is expressed: while by using an inducible or tissue-specific promoter, functions may be controlled more selectively in individual organs, tissues or cells, or at particular stages of the life cycle.
The present invention will find wide potential use in modifying plants in useful ways. Plant gene products that may be inhibited by the invention are of very diverse kinds. We believe that the mechanism of inhibition is independent of the nature of the gene or gene product. Thus in principle any the production of any kind of gene product may be inhibited.
Thus we propose to apply the process of the invention to the inhibition of gene products in all types of economically useful plants. These may include for example field crops such as corn (maize), sugar beet, sorghum, and sunflower; cereals such as wheat, barley and rice; legumes and pulses such as beans and peas; grasses; trees; leafy vegetable crops such as cabbage, lettuce and spinach; root crops such as potato, turnip, carrot; onions; fruit of all kinds; and ornamentals such as tulips, roses, carnations and azaleas.: The gene products to be inhibited may be those which are not desired in the crop (eg toxins) or those whose reduction can contribute to improved plant characteristics, such as yield, field performance or product quality.
By way of example only, and without any implied limitation on its field of use, we will describe the invention further with particular reference to possible uses in controlling fruit ripening processes.
The plants to which the present invention can be applied include commercially important fruit-bearing plants, for example melons, peaches, bananas, apples, strawberries, kiwi fruit, and in particular the tomato.
In this way, plants can be generated which may have one or more of the following characteristics:
Novel flavour and aroma due to changes in the concentrations and ratios of the many aromatic compounds that contribute to fruit flavour;
Sweeter fruit (e.g. tomatoes) due to decrease in the accumulation of acids (e.g. citric or malic acid) thereby allowing the flavour of the sugars to dominate;
Modified colour due to inhibition of the pathways of pigment biosynthesis (e.g. in the case of tomatoes, lycopene, &bgr;-carotene);
Longer shelf life and better storage characteristics due to reduced activity of degradative pathways (e.g. cell wall hydrolysis);
Improved processing characteristics due to changed activity of enzymes contributing to factors such as: viscosity, solids, pH, elasticity;
Modified fruit shape, thus improving packing and storage characteristics;
Extended leaf biosynthetic activity due to inhibition of enzymes responsible for the degradative processes involved in senescence (in particular, leaf senescence); thus improving plant productivity.
Among the gene products that c
Bridges Ian G.
Grierson Donald
Schuch Wolfgang W.
Nelson Amy J.
Pillsbury & Winthrop LLP
Zeneca Limited
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