Nucleic acid sequences coding for thanatin and transformed...

Details Nucleic acid sequences coding for thanatin and transformed... Nucleic acid sequences coding for thanatin and transformed...

2000-10-16

2004-08-03

McElwain, Elizabeth F. (Department: 1638)

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

The polynucleotide confers pathogen or pest resistance

C800S278000, C800S288000, C800S298000, C800S295000, C435S320100, C435S419000, C435S468000, C536S023700

Reexamination Certificate

active

06770798

ABSTRACT:

The subject of the present invention is a DNA sequence encoding thanatin, a vector containing it for the transformation of a host organism, and the method of transforming the said organism.
The invention relates more particularly to the transformation of plant cells and plants, the thanatin produced by the transformed plants conferring on them resistance to diseases, in particular of fungal origin.
An increasing need already exists for making plants resistant against diseases, in particular fungal diseases, in order to reduce, or even eliminate, the need for treatment with antifungal protection products, with a view to protecting the environment. One means of increasing this resistance to diseases consists in transforming the plants so that they produce substances capable of providing their defence against these diseases.
Various substances of natural origin, in particular peptides, are known which exhibit bactericidal or fungicidal properties, in particular against the fungi responsible for plant diseases. However, the problem consists in finding such substances which not only can be produced by transformed plants, but can still preserve their bactericidal or fungicidal properties and confer them on the said plants. For the purposes of the present invention, bactericidal or fungicidal is understood to mean the actual bactericidal or fungicidal properties and the bacteriostatic and fungistatic properties.
Thanatin is a peptide produced by bacterial induction on adult Psodius sp, preferably
maculiventris
. Its preparation by bacterial induction is described in patent application FR 2,733,237, as well as its antifungal and antibacterial properties in vitro.
After having first identified the thanatin gene, it was also found that it could be inserted into a host organism, in particular a plant, in order to express the thanatin and confer on the said host organism properties of resistance to fungal diseases and to diseases of bacterial origin, providing a particularly advantageous solution to the problem stated above.
The subject of the invention is therefore first a nucleic acid fragment encoding thanatin, a chimeric gene comprising the said fragment encoding thanatin as well as heterologous regulatory elements at the 5′ and 3′ positions which can function in a host organism, in particular in plants, and a vector for transforming the host organisms containing this chimeric gene, and the transformed host organism. It also relates to a transformed plant cell containing at least one nucleic acid fragment encoding thanatin and a disease-resistant plant containing the said cell, in particular regenerated from this cell. It finally relates to a method of transforming plants to make them resistant to diseases, in which method a gene encoding thanatin is inserted by means of an appropriate vector.
Thanatin is understood to mean according to the invention any peptide comprising essentially the peptide sequence of 11 amino acids which is described in patent application FR 2,733,237, as well as the equivalent homologous sequences in which certain amino acids are replaced by different but equivalent amino acids at sites which do not induce substantial modification of the antifungal or antibacterial activity of the said homologous sequence. Peptide sequence comprising essentially the peptide sequence described in patent application FR 2,733,237 is understood to mean not only the sequence defined by the sequence identifier No. 1 (SEQ ID NO 1), but also such a sequence comprising at either of its ends, or at both, peptide residues necessary for its expression and targeting in a host organism, in particular a plant cell or a plant.
Thanatin is a peptide of formula (I)(SEQ ID NO:14):
Xaa-Ile Ile Tyr Cys Asn Arg Arg Thr Gly Lys Cys-Xab  (I)
in which:
Xaa is NH
2
or a variable residue having a sequence comprising from 1 to 10 amino acids, and
Xab is OH or a variable residue having a sequence comprising from 0 to 5 amino acids.
Advantageously, when Xaa comprises at least one amino acid, the latter is one of the 20 base amino acids and more particularly chosen from the group comprising Gly, Ser, Lys, Pro and Val. When Xab comprises at least one amino acid, the latter is one of the 20 base amino acids and more particularly chosen from the group comprising Gln, Arg and Met.
According to a preferred embodiment of the invention, the two cysteine residues of the peptide of formula (I) form an intramolecular disulphide bridge.
The present invention therefore relates first to a nucleic acid, in particular a DNA, fragment encoding the thanatin defined above. It may be, according to the invention, a fragment isolated from Psodius sp, preferably
maculiventris
, or alternatively a derived fragment, suitable for the expression of thanatin in the host organism where the peptide will be expressed. The nucleic acid fragment may be obtained using standard methods of isolation and purification, or alternatively by synthesis according to the customary techniques of successive hybridizations of synthetic oligonucleotides. These techniques are in particular described by Ausubel et al.
According to the present invention, “nucleic acid fragment” is understood to mean a nucleotide sequence which may be of the DNA or RNA type, preferably of the DNA, in particular cDNA, especially double-stranded, type.
According to one embodiment of the invention, the nucleic acid fragment encoding thanatin comprises the DNA sequence described by the sequence identifier No. 1 (SEQ ID NO 1), a homologous sequence or a sequence complementary to the said sequence.
Advantageously, the nucleic acid fragment according to the invention comprises the DNA sequence described by the sequence identifier No. 2 (SEQ ID NO 2), a homologous sequence or a sequence complementary to the said sequence.
“Homologous” is understood to mean according to the invention a nucleic acid fragment having one or more sequence modifications relative to the nucleotide sequence described by the sequence identifier No. 1 or No. 2 and encoding thanatin. These modifications may be obtained according to the customary mutation techniques, or alternatively by choosing the synthetic oligonucleotides used in the preparation of the said sequence by hybridization. Given the multiple combinations of nucleic acids which may lead to the expression of the same amino acid, the differences between the reference sequence described by the sequence identifier No. 1 or No. 2 and the homologue may be great, especially since a DNA fragment of less than 100 nucleic acids in size, which can be produced by synthesis, is involved. Advantageously, the degree of homology will be at least 70% relative to the reference sequence, preferably at least 80%, more preferably at least 90%. These modifications are generally neutral, that is to say that they do not affect the primary sequence of the resulting thanatin.
The present invention also relates to a chimeric gene (or an expression cassette) comprising a coding sequence as well as heterologous regulatory elements at the 5′ and 3′ positions which can function in a host organism, in particular plant cells or plants, the coding sequence comprising at least one DNA fragment encoding thanatin as defined above.
Host organism is understood to mean any higher or lower mono- or pluricellular organism into which the chimeric gene according to the invention may, be introduced, for the production of thanatin. It consists of in particular bacteria, for example
E. coli
, yeasts, in particular of the genera Saccharomyces or Kluyveromyces, or preferably plant cells and plants. “Plant cell” is understood to mean according to the invention any cell derived from a plant and which may constitute undifferentiated tissues such as calli, and differentiated tissues such as embryos, plant portions, plants or seeds.
“Plant” is understood to mean according to the invention any differentiated multicellular organism capable of photosynthesis, in particular monocotyledones or dicotyledones, more particularly cultivated plants intended or otherwise

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