Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1998-10-27
2001-02-27
Fox, David T. (Department: 1638)
Chemistry: molecular biology and microbiology
Vector, per se
C435S069100, C435S468000, C435S206000, C536S024100, C800S278000, C800S301000, C800S302000, C800S317200, C800S320200, C800S320100, C800S306000, C800S312000, C800S313000, C800S314000, C800S317400, C800S279000, C800S283000, C800S284000, C800S288000
Reexamination Certificate
active
06194201
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to an expression system for anaerobic gene expression in higher plants. A concrete field of application of the present invention is agriculture, particularly resistance cultivation and the increase in the efficiency of useful plants.
BACKGROUND OF THE INVENTION
The loss of harvested crops, which results from diseases of plants, represents a world-wide problem. For example, potatoes suffering from potato wet rot or potato rot (rotting of the tuber) and black leg or black stem (rotting of the lower stem sections) after infection by the phytopathogenic bacterium
Erwinia carotovora
, results in crop losses to an estimated amount of 100 million dollars world-wide (Pérombelon et al., 1980,
Ann. Rev. Phytopathol.
18: 361-387). There is a number of studies dealing with the transmission of resistance factors to plants by means of genetic engineering (Lamb et al., 1992
, Bio/Technology
10: 1436-1445; Hain et al., 1994,
Current Opinion in Biotechnology,
125-130; Zhu et al., 1994,
Bio/Technology
12: 807-812). In order to increase the resistance of potatoes to
Erwinia carotovora,
the T4 lysozyme gene of the bacteriophage T4 was expressed in transgenic potatoes (Düring et al., 1993,
Plant J.
3: 587-598).
However, since bacterial diseases of plants often spread under anaerobic conditions, the resistance factors for plants transmitted so far, are effective only in a very restricted extent. This applies particularly to the above-mentioned disease of potatoes suffering from potato rot and black leg, since the infection caused by
Erwinia carotovora
takes place predominantly under anaerobic conditions. This effect is increased by the formation of a mucus from bacteria and degradation products of vegetable cell membranes. Regarding an effective expression of an antibacterial protein under optimum conditions, it is desirable to control the corresponding foreign gene by a promoter active under these conditions.
Three anaerobic promoters have been tested in transgenic plants so far. These are the Adh1 promoter from corn, the Adh promoter from
Arabidopsis thaliana
and the GapC promoter from
Arabidopsis thaliana.
The Adh1 promoter from corn was investigated in tobacco and rice (Ellis et al., 1987,
EMBO J.
6: 11-16; Kyozuka et al., 1991,
Mol. Gen. Genet.
228: 40-48). The GapC promoter from Arabidopsis was investigated in tobacco (Yang et al., 1993,
Plant Physiol.
101: 209-210), and the Adh promoter from Arabidopsis was investigated in Arabidopsis as such (Dolferus et al., 1994,
Plant Physiol.
105: 1075-1087). It turned out that all promoters convey only 2 to 81 times the induction of the reporter gene over the background and are not active in all of the tissues.
SUMMARY OF THE INVENTION
One objective of this invention to achieve the expression of resistance factors in useful plants and to produce corresponding transgenic plants. It is a further object of this invention to achieve the anaerobic expression of the T4 lysozyme gene in potatoes and produce corresponding transgenic plants. A specific object consists in making potatoes resistant to phytopathogenic bacteria.
The present invention is achieved by an expression system comprising the GapC4 promoter or parts or variants of the GapC4 promoter and a gene to be expressed.
DETAILED DESCRIPTION OF THE INVENTION
According to the invention, genes for an antibacterial protein, particularly for a T4 lysozyme, resistance genes (r-determinants) against viruses, nematodes, bacteria and fungi, genes having an insecticidal effect, glycolysis-increasing genes and fermentation-increasing genes are provided.
According to the invention, the expression system is applied to an anaerobic gene expression in higher plants. It is used preferably in cultivated plants such as potatoes, rice, grain, corn, tomatoes, brassicaceae, leguminous plants, cotton, sugar beets and carrots.
This invention also relates to higher plants, preferably transgenic cultivated plants such as potatoes, rice, grain, corn, tomatoes, brassicaceae, leguminous plants, cotton, sugar beets and carrots, which contain the expression system according to the invention.
Transgenic potatoes that contain an expression system from a GapC4 promoter and the gene for a T4 lysozyme are of special importance.
The major advantage of the GapC4 promoter used according to the invention (GenBank accession No. L40803) consists in that it has an induction profile excellently suited for the objective. The anaerobic expression achieves the intensity of the 35S promoter of cauliflower mosaic virus (35S CaMV) which is frequently used under aerobic conditions for the expression of foreign genes. Furthermore, the promoter is active in all tissues such as the flower or blossom, leaf and root. The anaerobic induction of the GapC4 promoter has not been known neither in plants nor in a transient expression system. Surprisingly, the promoter is especially active in cultivated potatoes.
Additionally, there are other potential applications of the invention. Another problem is represented, especially in moist climate, by periodic flooding of fields, which may lead to crop failure. As they are aerobic organisms, plants cannot survive prolonged periods of extensive moisture which results in a decrease of oxygen available to the plants (Perata and Alpi, 1993, Plant Sci. 93, 1-17). The tolerance of plants towards insufficient oxygen supply differs rather widely for individual species. For example, the embryo in rice calyopsis also germinates under these conditions without any difficulties, whereas corn germs survive without oxygen for only about 24 hours. A general adaptive strategy of higher plants to anaerobic conditions is the increase in glycolysis as well as the starting of fermentation processes. In order to increase the tolerance of plants towards insufficient oxygen supply, the genes which take part in glycolysis as well as fermentation can be controlled by an anaerobically inducible promoter. These genes are then expressed in the case of insufficient oxygen supply.
The present invention is explained in more detail below by way of the examples. However, the intention is not intended to be limited thereby as the examples are merely illustrative.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
REFERENCES:
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Dolferus, et al., “Differential Interactions of Promoter Elements in Stress Responses of theArabidopsis AdhGene,”Plant Physiol.—105:1075-1087 (1994).
Düring, et al., “Transgenic Potato Plants Resistant to the Phytopathogenic BacteriumErwinia Carotovora,” The Plant Journal—3(4):587-598 (1993).
Ellis, et al., “Maize Adh-1 Promoter Sequences Control Anaerobic Regulation: Addition of Upstream Promoter Elements from Constitutive Genes is Necessary for Expression in Tobacco,”The EMBO Journal—6(1):11-16 (1987).
Fischer, et al., “Plant Disease Resistance Resulting from the Expression of Foreign Phytoalexins,”Current Opinion in Biotechnology—5:125-130 (1994).
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Honma, et al., “High-Frequency Germin
Cerff Rüdiger
Düring Klaus
Hehl Reinhard
Kohler Uwe
Fox David T.
Halluin Albert T.
Howrey Simon Arnold & White , LLP
Kung Viola T.
Mehta Ashwin D.
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