Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1995-05-23
2001-05-22
Fox, David T. (Department: 1638)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S069200, C435S469000, C435S419000, C435S069800, C800S301000, C800S306000, C800S278000
Reexamination Certificate
active
06235530
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The subject of the present invention is a gene encoding an oxalate oxidase, the protein encoded by this gene, the chimeric genes comprising this gene and their use for transformation of dicotyledonous plants in order to confer on those plants a resistance to fungal diseases.
2. Description of the Related Art
Sclerotiniosis is a major fungal disease which affects a large number of dicotyledons. The causative agent,
Sclerotinia sclerotiorum
is a polyphagous fungus which exhibits little host specificity.
The fungus can attack the plant either directly at the level of the stem, or at the level of the leaves and then spread to the stem, or at the level of the floral capitulum. In the first two cases, the plant withers from disruption to food supply. In the last case, the flower withers, damaging the harvest.
The fungus produces lytic enzymes which degrade the cell wall of the infected plant and promote its development in the plant. These enzymes play an important role in pathogenicity, but do not appear to be sufficient. This fungus also produces oxalic acid (Godoy et al. (1990).
Physiol. Molec. Plant. Pathol).
37:179-181. This oxalic acid causes a decrease in pH in the infected tissues, promoting hydrolysis of the cell wall by the lytic enzymes. A reduction in the production of oxalic acid or degradation of this oxalic acid should permit a slowing-down or even an inhibition of the development of the fungus.
In order to develop a Sclerotinia resistant plant, the strategy of detoxification of oxalic acid may be used. The degradation of this acid will limit the decrease in intracellular pH of the plant tissue attacked, the lytic enzymes will thereby be functioning at a value too far-removed from their optimum pH to be really active and efficient. This will lead to a decrease in the pathogenicity of the fungus.
Oxalate oxidase which catalyses the following reaction:
C
2
O
4
H
2
--
-
--
--
--
->
O
2
oxalate
oxidase
2
CO
2
+
H
2
O
2
may be used to achieve this objective.
Oxalate oxidase is isolated from various plants, generally from monocotyledons (Pieta et al. (1982)
Preparative Biochemistry
12(4):341-353): the protein may for example be purified from barley using conventional chromatographic techniques (Sephadex G-75 filtration gels and MonoQ ion exchange gels, Pharmacia), by monitoring the enzymatic activity according to the following calorimetric procedure (Obzansky and Richardson (1983)
Clin. Chem.
29(10):1815-1819):
This has made it possible to purify a protein which, on acrylamide gel under denaturing conditions, has a molecular mass of 26,000 daltons. Part of the purified oxalate oxidase was used to obtain rabbit anti-oxalate oxidase antibodies; the remainder of the protein was used to carry out the sequencing of the native protein (N-terminal) or, after cyanogen bromide cleavage, the sequencing of certain internal peptides. The results obtained are as follows (SEQ ID NOS:1-2):
N-terminal: SIDPDPLQDF-VADLDGKAVSVNGH
Internal peptide No. 2: HFQFNVGKTEAY cDNA
Comparison of the peptide sequences described above with the data contained in the protein library Swiss-Prot enabled us to identify a wheat protein called Germine and published in 1989 by Dratewka-kos et al. Experiments were carried out and they enabled us to determine that the cDNA published by the authors encodes a protein of 201 amino acids which exhibits an oxalate oxidase activity. For the rest of the description of the experiments presented in this patent, we will use the nucleotide numbering in
FIG. 2
in the article by the authors published in J. Biol. Chem., 264, 4896-4900. This sequence is reproduced as
FIG. 2
of the present application.
The sequence of this cDNA is 1075 nucleotides in length with an untranslated 5′ of 85 residues, an open reading frame of 672 nucleotides (from position 86 to 757) and an untranslated 3′ of 318 residues.
Comparison of the protein sequence deduced from the cDNA sequence with that obtained by sequencing the native protein shows that the cDNA encodes not only mature oxalate oxidase but also a signal peptide of 23 amino acids in the N-terminal part. Oxalate oxidase is therefore synthesised in the form of a preprotein (signal peptide plus mature peptide) which undergoes maturation by removal of the signal peptide in order to release the mature active enzyme.
REFERENCES:
patent: 5866778 (1999-02-01), Hartman et al.
Dratewka-Kos et al., “Polypeptide Structure of Germin as Deduced from cDNA Sequencing,”The Journal of Biological Chemistry(1989) 264(9):4896-4900.
Lane et al., “Homologies between Members of the Germain Gene Family in Hexaploid Wheat and Similarities between These Wheat Germins and Certain Physarum Spherulins,”The Journal of Biological Chemistry(1991) 266(16):10461-10469.
Freyssinet Georges
Sailland Alain
Burns Doane Swecker & Mathis L.L.P.
Fox David T.
Kimball Melissa L.
Rhone-Poulenc Agrochimie
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