HRP980455A2 - Gene coding for androctonine, vector containing it and disease-resistant transformed plants obtained - Google Patents

Description

The present invention relates to a DNA sequence coded for androctonin, a vector for the transformation of a host organism that contains it, and a process for transforming said organism.

The invention particularly relates to the transformation of plant cells and plants, and to androctonin produced by transformed plants, whereby resistance to diseases, primarily diseases of fungal origin, is created.

Today, there is an increasing need to obtain disease-resistant plants, especially fungal diseases, in order to reduce, even completely avoid treatments with anti-fungal protection products, with the aim of protecting the environment. One of the ways to increase this resistance to diseases consists in transforming plants, so that they produce substances that by themselves enable their defense against diseases.

Different substances of natural origin are known, primarily peptides that show bactericidal or fungicidal properties, especially against fungi responsible for plant diseases. However, the problem is finding those substances that are not only produced by transformed plants, but also retain their bactericidal or fungicidal properties, and give them to said plants. In the sense of the present invention, the terms bactericidal or fungicidal refer both to actual bactericidal or fungicidal properties, as well as to bacteriostatic and fungistatic properties.

Androctonins are peptides produced by scorpions, primarily those of the species Androctonus Australis. One androctonin and its preparation by chemical synthesis were described by Ehret-Sabatier et al., as well as its in vitro antifungal and antibacterial properties.

Androctonin genes have now been identified, and it has also been found that they can be inserted into a host organism, primarily into a plant, with the aim of expressing androctonin both for the preparation and isolation of that androctonin, and for conferring on said host organism the property of resistance to fungal diseases and diseases of bacterial origin , which provides a particularly suitable solution to the above-mentioned problem.

Therefore, the subject of the invention is primarily a nucleic acid fragment encoded for some androctonin, a chimeric gene containing said fragment encoded for some androctonin, then heterologous regulatory elements at the 5' and 3' positions that can function in the host organism, primarily in plants, and a vector for the transformation of organisms the host containing that chimeric gene as well as the transformed host organism. The invention also relates to a transformed plant cell containing at least one nucleic acid fragment encoded for androctonin, and a disease-resistant plant containing said cell. Finally, the invention also relates to the process of growing transformed plants according to the invention.

According to the invention, the term androctonin refers to any peptide that can be produced by scorpions or that can be isolated from them, preferably from the species Androctonus australis, wherein these peptides contain at least 20 amino acids, preferably at least 25, and 4 cysteine residues that together form disulfide bridges.

It is advantageous that androctonin essentially contains a peptide sequence of the following general formula (I):

Xaa-Cys-Xab-Cys-Xac-Cys-Xad-Cys-Xae (I)

where

Xaa represents a peptide residue containing at least 1 amino acid,

Xab represents a peptide residue of 5 amino acids,

Xac represents a peptide residue of 5 amino acids,

Xad represents a peptide residue of 3 amino acids, a

Xae represents a peptide residue containing at least 1 amino acid.

Advantageously, Xab and/or Xad and/or Xae contain at least one basic amino acid, preferably 1. According to the invention, the term basic amino acid means an amino acid selected from lysine, asparagine and homoasparagine.

First of all,

Xaa represents the peptide sequence Xaa'-Val, in which

Xaa' represents NH2 or a peptide residue containing at least 1 amino acid, and/or

Xab represents the peptide sequence -Arg-Xab'-Ile, in which

Xab' represents a peptide residue of 3 amino acids, and/or

Xac represents the peptide sequence -Arg-Xac'-Gly-, in which

Xac' represents a peptide residue of 3 amino acids, and/or

Xad represents the peptide sequence -Tyr-Xad'-Lys, in which

Xad' represents a peptide residue of 1 amino acid, and/or

Xae represents the peptide sequence -Thr-Xae', in which

Xae' represents COOH or a peptide residue containing at least 1 amino acid.

First of all,

Xaa' represents the peptide sequence Arg-Ser-, and/or

Xab' represents the peptide sequence -Gln-Ile-Lys-, and/or

Xac' represents the peptide sequence -Arg-Arg-Gly-, and/or

Xad' represents the peptide residue -Tyr-, and/or

Xae' represents the peptide sequence -Asn-Arg-Pro-Tyr.

According to the most favorable form of the invention, androctonin is represented by a peptide sequence of 25 amino acids, described by sequence identifier no. 1 (SEQ ID No 1) and homologous peptide sequences.

Homologous peptide sequence means all equivalent sequences that are at least 65% homologous with the sequence represented by sequence identifier no. 1, whereby it is understood that the 4 cysteine residues and the series of amino acids that separate them remain identical, while some amino acids are replaced by different but equivalent ones, in positions that do not induce significant changes regarding the antifungal or antibacterial activity of the mentioned homologous sequence. Primarily homologous sequences contain at least 75% homology, especially at least 85% homology, and above all 90% homology.

The NH2-terminal residue of androctonin can show post-translational modification, for example acetylation, while the C-terminal residue can show post-translational modification, for example amidation.

An expression peptide sequence that essentially contains a peptide sequence of the general formula (I) means not only the above-mentioned sequences, but also all those sequences that contain at one or the other end, or at both ends, peptide residues, especially those necessary for their expression and targeting in host organisms, primarily in plant cells or plants.

This primarily refers to "peptide-androctonin" or "androctonin-peptide", primarily "peptide-androctonin", the cleavage of which in the enzymatic systems of plant cells allows the release of androctonin as defined below. The peptide linked to androctonin can be a signal peptide or a transit peptide that allows the control and direction of androctonin production in a specific way in a part of the plant cell or plant, such as for example the cytoplasm or the cell membrane, or in the case of plants in a specific type of cell compartment or tissue or in extracellular matrix.

In one embodiment, the transit peptide can be a chloroplast or mitochondrial targeting signal, which is then cleaved in the chloroplast or mitochondrion.

According to another aspect of the invention, the signal peptide can be an N-terminal signal or "prepeptide", optionally linked to a signal responsible for the retention of the protein in the endoplasmic reticulum, or a vacuolar targeting peptide or "propeptide". The endoplasmic reticulum is the place where the "cellular machine shop" takes over the maturation operations of the produced protein, such as cleavage of the signal peptide.

Transit peptides can be either single or double, in which case they are optionally separated by an intermediate sequence, that is, one that contains in the direction of transcription the sequence coded for the transit peptide of the plant gene that is coded for the plastid localization enzyme, part of the sequence of the mature N-terminal part of the plant gene coded for a plastid localization enzyme, and a sequence coded for another transit peptide of a plant gene that is coded for a plastid localization enzyme, as described in patent application EP 0 508 909.

As a transit peptide useful according to the invention, the signal peptide of the tobacco PR-1α gene (WO 95/19443), represented by its coded sequence using the sequence identifier no. 2 (SEQ ID NO.2), and fused with androctonin using sequence identifier no. 3 (SEQ ID NO. 3), preferably in the corresponding fusion peptide corresponding to bases 12 to 176 of that sequence, and especially when androctonin is produced in plant cells or plants, or the precursor of the Mat α1 factor when androctonin is produced in yeasts.

Therefore, the present invention relates primarily to a nucleic acid fragment, primarily a DNA fragment, coded for androctonin as defined above. According to the invention, it can be a fragment isolated from Androctonus australis, or an alternatively derived fragment adapted for the expression of androctonin in the host organism in which the peptide will be expressed. The nucleic acid fragment can be obtained according to standard methods for isolation and purification, or alternatively by synthesis according to conventional techniques of sequential hybridization of synthetic oligonucleotides. These techniques were first described by Ausubel et al.

According to the present invention, the term "nucleic acid fragment" refers to a nucleotide sequence that can be either of the DNA type or the RNA type, preferably of the DNA type, and primarily of the cDNA, preferably of the double-stranded type.

According to the invention, the nuleic acid fragment coded for androctonin is the DNA sequence described by the sequence identifier no. 1 (SEQ ID NO. 1), a homologous sequence or a sequence complementary to said sequence, preferably encoding a part of it SEQ ID NO. 1, which corresponds to bases 1 to 75.

According to the invention, the term "homologous" refers to nucleic acid fragments that include one or more modifications compared to the nucleotide sequence described by sequence identifier no. 1 coded for androctonin. These modifications can be obtained by conventional mutation techniques or alternatively by selecting synthetic oligonucleotides used in the preparation of said sequence by hybridization. Given the multiple combinations of nucleic acids that can lead to the expression of the same amino acid, the differences between the reference sequence described by sequence identifier no. 1 and homologous sequences can be substantial, especially if it is a DNA fragment whose size is less than 100 nucleic acids, and which can be prepared by chemical synthesis. Preferably, the degree of homology to the reference sequence is at least 70%, preferably at least 80%, and most preferably at least 90%. These modifications are generally neutral, that is, they do not affect the primary sequence of the resulting androctonin.

The present invention also relates to a chimeric gene (or expression cassette) containing a coding sequence and heterologous regulatory elements in the 5' and 3' positions that can function in a host organism, preferably in plant cells or plants, wherein these elements are functionally linked to said coding sequence, and the coding sequence comprises at least one DNA fragment coding for androctonin as defined above (including a “peptide-androctonin” or androctonin-peptide fusion peptide”).

It is understood that the term host organism refers to any unicellular or multicellular organism of lower order or higher order, into which the chimeric gene according to the invention can be introduced, with the aim of producing androctonin. Such organisms are primarily E. coli, yeasts, primarily yeasts of the genus Saccharomyces or Kluyveromyces, Pichia, fungi, primarily Aspergillus, baculovirus or primarily plant cells and plants.

According to the invention, "plant cell" means all cells originating from a single plant that can form undifferentiated tissues such as calluses, differentiated tissues such as embryos, plant parts, plants or sprouts.

According to the invention, the term "plant" means all differentiated multicellular organisms capable of carrying out photosynthesis, primarily monocotyledons or dicotyledons, and especially plant crops intended for feeding animals or humans, or without such a purpose, such as corn, grain, rapeseed, soy, rice, sugar cane, sugar beet, tobacco, cotton, etc.

The regulatory elements required for the expression of the DNA fragment coding for androctonin are well known to those skilled in the art as a function of the host organism. They primarily include promoter sequences, transcriptional activators, transit peptides and termination sequences, and start and stop codons. Means and methods of identification and selection of regulatory elements are well known to a person skilled in the art.

For the transformation of microorganisms such as yeasts or bacteria, regulatory elements are well known to those skilled in the art, and primarily include promoter sequences, transcriptional activators, transit peptides, termination sequences and start and stop codons.

In order to direct the expression and secretion of the peptide in the yeast culture medium, the DNA fragment coded for heliomycin was incorporated into a transfer vector containing the following elements:

• markers that enable the selection of transformers,

• a nucleic acid sequence that enables the replication (origin of replication) of a plasmid in yeast,

• the nucleic acid sequence that enables the replication (origin of replication) of the plasmid in E. coli,

• an expression cassette consisting of

1. promoter regulatory sequences,

1. sequences coded for a signal peptide (or prepeptide) combined with an addressing peptide (or propeptide),

1. polyadenylation or terminator regulatory sequences.

These elements are described in several publications, including Reichhart et al., Invert. Reprod. Dev., 21 (1992) 15-24, and Michaut et al., FEBS Letters, 395 (1996) 6-10.

In particular, yeasts of the S. cerevisiae species are transformed using expression plasmids by the lithium acetate method (Ito et al., J. Bacteriol. 153 (1993) 163-168).

The invention relates in particular to the transformation of plants. As a promoter regulatory sequence in plants, it is possible to use any gene promoter sequence that is naturally expressed in plants, primarily a promoter of bacterial, viral or plant origin, such as that of the gene for the small subunit of ribulose biscarboxylase/oxygenase (RuBisCO), or that of plant a viral gene such as for example that of cauliflower mosaic virus (CAMV 19S or 35S), or a pathogen inducible promoter such as tobacco PR-1α, where all known suitable promoters can be used. Preferably, a promoter regulatory sequence is used which favors the overexpression of the encoded sequence in a constitutive manner or induced by pathogen attack, such as for example one containing at least one histone promoter as described in patent application EP 0 507 698.

According to the invention, it is also possible to apply, together with the promoter regulatory sequence, other regulatory sequences, which are located between the promoter and the coding sequence, such as transcriptional activators ("enhancers"), for example the translational activator of the tobacco mosaic virus (TMV) described in the patent application WO 87/07644, or the tobacco etch virus (TEV) translational activator described by Carrington & Freed.

Any appropriate sequence of bacterial origin, such as the Agrobacterium tumefaciens terminator, or alternatively of plant origin, such as the histone terminator described in patent application EP 0 633 317, can be used as a polyadenylation or termination regulatory sequence.

According to the present invention, the chimeric gene can be combined with a selection marker adapted to the transformed host organism. Such selectable markers are well known to those skilled in the art. Such markers can be an antibiotic resistance gene or a herbicide tolerance gene for plants.

The present invention also relates to a clonal or expression vector for transformation of a host organism containing at least one chimeric gene as defined above. In addition to the above chimeric gene, this vector contains at least one origin of replication and, if necessary, a suitable selection marker. This vector can consist of a plasmid, cosmid, bacteriophage or virus, which have been transformed by introducing a chimeric gene according to the invention. Depending on the host organism to be transformed, such transformation vectors are well known to those skilled in the art and are described in detail in the literature.

For the transformation of plant cells or plants, such a vector is primarily a virus that can be used for the transformation of developed plants, which also contain their own replication and expression elements. First of all, the vector for the transformation of plant cells or plants according to the invention is a plasmid.

The subject of the invention is also the process of transformation of the host organism, primarily plant cells, by incorporating at least one nucleic acid fragment or one chimeric gene according to the above definition, whereby it is possible to achieve this transformation by any of the known suitable methods that are sufficiently described in the specialized literature, and in particular in the statements cited in the present patent application, primarily by means of the vector according to the invention.

A number of methods consist of bombarding cells, protoplasts or tissues with particles to which DNA sequences are attached. Another series of methods consists in the application of a chimeric gene inserted into the Ti plasmid from Agrobacterium tumefaciens or the Ri plasmid from Agrobacterium rhizogenes as a means of transfer into the plant.

Among other methods, microinjection or electroporation methods can be used, or alternatively, direct deposition using PEG.

An expert in the field will select a suitable method according to the nature of the host organism, preferably a plant cell or plant.

The subject of the present invention is also transformed host organisms, in particular plant cells or plants, which contain an effective amount of a chimeric gene including the sequence coding for androctonin as defined above.

Also subject to the present invention are plants containing transformed cells, especially plants regenerated from transformed cells. Regeneration is achieved by any suitable process depending on the nature of the species, as described for example in the above references.

For plant cell transformation and plant regeneration procedures, the following patents and patent applications should first be cited: US 4,459,355, US 4,536,475, US 5,464,763, US 5,177,010, US 5,187,073, EP 267 159, EP 604 662, EP 672 752, US 4,945,050 US 5,100,792, US 5,371,014, US 5,478,744, US 5,179,022, US 5,565,346, US 5,484,956, US 5,508,468, US 5,538,877, US 5,554,798, US 5,489,520, US 5,510,318, US 5,204,253, US 5,405,765, EP 442 174, EP 486 233, EP 486 234 , EP 539 563, EP 674 725, WO 91/02071 and WO 95/06128.

The subject of the present invention are also transformed plants obtained by growing and/or crossing regenerated plants, as well as seeds of transformed plants.

Plants transformed in this way are resistant to some diseases, especially some fungal or bacterial diseases. Consequently, the DNA sequence coding for androctonin can be inserted with the main means of producing plants resistant to said diseases, since androctonin is effective against fungal diseases such as those caused by Cerospora species, in particular Cerospora beticola, Cladosporium, in particular Cladosporium herbarum, Fusarium, primarily Fusarium culmorum or Fusarium graminearum, or Phytophtora, primarily Phytophtora cinnamoni.

The chimeric gene can preferably be combined with at least one selection marker, such as one or more herbicide tolerance genes.

The DNA sequence coding for androctonin can also be inserted as a selection marker during transformation of plants with other sequences coding for other peptides or proteins of interest, such as for example herbicide tolerance genes.

Such herbicide tolerance genes are well known to those skilled in the art and are particularly described in patent applications EP 115 673, WO 87/04181, EP 337 899, WO 96/38567 or WO 97/04103.

Of course, cells and plants transformed according to the invention may also contain a sequence coding for androctonin, other heterologous sequences coding for proteins of interest such as other complementary peptides capable of conferring resistance to plants against other diseases of bacterial origin or fungal origin, and/or other sequences coding for for herbicide tolerance proteins, preferably those defined earlier, and/or other sequences encoded for insect resistance proteins, such as preferably the Bt protein.

Other sequences can be inserted using the same vector containing the chimeric gene according to the invention, which contains the sequence coding for androctonin and at least one other gene which includes another sequence coding for some other peptide or protein of interest.

They may also be inserted by means of another vector containing at least one of said other sequences, according to the usual techniques mentioned above.

Plants according to the invention can also be obtained by crossing parents, one of which carries a gene according to the invention encoding androctonin, and the other carries a gene encoding at least one other peptide or protein of interest.

Among the sequences encoding other antifungal peptides, one can mention that encoding drosomycin, described in patent application FR 2,725,992 and described by Fehlbaum et al. (1994), and in unpublished patent application FR 97/09115 received July 24, 1997.

The present invention finally relates to the process of growing plants transformed according to the invention, a process that consists of sowing the seeds of said transformed plants in the surface of the growing environment, primarily a field suitable for growing said plant, applying an agrochemical preparation to said surface, without significant impact on said transformed seeds or plants, then harvesting the cultivated plants that have reached the desired maturity and optionally separating the seeds from the harvested plants.

According to the invention, the term agrochemical preparations means all agrochemical preparations that contain at least one effective product that has either herbicidal, fungicidal, bactericidal, virucidal or insecticidal activity.

According to the method of realization of the cultivation process which is advantageous according to the invention, the agrochemical preparation contains at least one effective product which shows at least fungicidal and/or bactericidal activity, and in addition, it primarily possesses an activity complementary to that of androctonin produced in the plant transformed according to the invention.

According to the invention, an expression product that has an activity complementary to that of androctonin means a product that possesses a spectrum of complementary activities, which means a product that will be active against the attack of contaminants insensitive to androctonin (fungi, bacteria or viruses), or alternatively a product whose spectrum of activity completely or it partially covers that of androctonin, and whose application dose is significantly reduced due to the presence of androctonin produced in the transformed plant.

Finally, cultivation of transformed host organisms allows large-scale production of androctonin. The subject of the present invention is therefore also a process for the preparation of androctonin, which includes the steps of growing transformed host organisms containing the gene encoded for androctonin as defined above in a suitable culture environment, and then extracting and completely or partially purifying the obtained androctonin.

The following examples illustrate the invention, the preparation of the sequence coded for androctonin, the chimeric gene, the integration vector and the transformed plants. Attached figures 1 to 5 outline the schematic structures of some plasmids prepared for the construction of chimeric genes. In these figures, different restriction sites are marked in italics.

Example 1

Construction of chimeric genes

All further applied techniques are standard laboratory techniques. The detailed procedures of these techniques were particularly described by Ausubel et al.

pRPA-MD-P: Preparation of a plasmid containing a signal peptide for the tobacco PR-1α gene.

Two complementary synthetic oligonucleotides, Oligo 1 and Oligo 2, listed below, were hybridized at 65 °C for 5 minutes, and then slowly lowering the temperature to 30 °C for 30 minutes.

[image]

After hybridization between Oligo 1 and Oligo 2, the remaining single-stranded DNA serves as a template for the klenow fragment from E. coli polymerase 1 (under standard conditions recommended by the manufacturer (New England Biolabs)) to prepare a double-stranded oligonucleotide starting from the 3' end of each oligo. The resulting double-stranded oligonucleotide was then digested with restriction enzymes SacII and NaeI and cloned into plasmid pBS II SK(-) (Stratagene) digested with the same restriction enzymes. A clone was obtained that contained the region coded for the signal peptide of the tobacco PR-1α gene (SEQ ID NO. 2).

pRPA-PS-PR1a-andro: Preparation of the sequence coding for androctonin fused to the PR-1α signal peptide without the non-transcribed 3' region.

Two complementary synthetic oligonucleotides Oligo 3 and Oligo 4 were hybridized according to the operating conditions specified for pRPA-MD-P.

[image]

After hybridization between Oligo 3 and Oligo 4, the remaining single-stranded DNA serves as a template for the klenow fragment from E. coli polymerase 1 (under standard conditions recommended by the manufacturer (New England Biolabs)) to prepare a double-stranded oligonucleotide starting from the 3' end of each oligo. The obtained double-stranded oligonucleotide containing the part encoded for androctonin (SEQ ID NO. 1) was then cloned directly into the plasmid pRPA-MD-P, which was digested with the restriction enzyme NaeI. The correct orientation of the obtained clone was verified by sequencing. This resulted in a clone containing the region coded for the fusion protein PR-1α-androctonin, located between the restriction site at the N-terminal end and the restriction sites ScaI, ScaII and BamHI at the C-terminal end (SEQ ID NO. 3).

pRPA-RD-238: Preparation of an expression vector in plants containing the sequence coded for the fusion protein PR-1α androctonin.

Plasmid pRTL-2 GUS derived from plasmid pUC-19 was obtained from Dr. Jim Carrington (Texas A&M University, undescribed). This plasmid, the schematic structure of which is shown in Figure 3, contains a CaMV 35S double promoter isolated from cauliflower mosaic virus (CaMV 2x35S promoter; Odell et al., 1985) that directs the expression of an RNA containing the 5' untranslated sequence of tobacco etch virus (TEV). 5' UTR; Carrington and Freed, 1990), the β-glucuronidase gene from E. coli (GUS; Jefferson et al., 1987), then the CaMV RNA 35S polyadenylation site (CaMV polyA; Odell et al., 1985).

Plasmid pRTL-2 GUS was digested with restriction enzymes NcoI and BamHI and the main DNA fragment was purified. Plasmid pRPA-PS-Pr1a-andro was digested with restriction enzymes NcoI and BamHI and a small DNA fragment containing the region coding for the fusion protein PR-1a-androctonin was also purified. The two purified DNA fragments were then joined together in an expression cassette in plants that synthesize the PR-1a-androctonin fusion protein. The schematic structure of these expression cassettes is shown in Figure 2. “PR-1a-androctonin” represents the region encoded for the PR-1a-androctonin fusion protein from pRPA-RD-230. Androctonin is transported into the extracellular matrix of the plant by the action of the signal peptide PR-1a.

pRPA-RD-195: Preparation of a plasmid containing a modified multiple cloning site.

Plasmid pRPA-RD-195 is a plasmid derived from pUC-19 containing a modified multiple cloning site. The complementary synthetic oligonucleotides Oligo 5 and Oligo 6 shown below were hybridized and double-stranded according to the procedure described for pRPA-MD-P.

[image]

The resulting double-stranded oligonucleotide was then inserted into pUC-19, which was previously digested with the restriction enzymes EcoRI and HindIII and blunted at the ends using a fragment of klenow DNA polymerase 1 from E. coli. A vector containing multiple cloning sites was obtained to enable the introduction of expression cassettes into a plasmid vector from Agrobacterium tumefaciens. The schematic structure of the multiple clone position is shown in Figure 3.

pRPA-RD-233: Introduction of the PR-1a-androctonin expression cassette from pRPA-RD-230 into pRPA-RD-195.

Plasmid pRPA-RD-230 was digested with the restriction enzyme HindIII. The DNA fragment containing the PR-1a-androctonin expression cassette was purified. The purified fragment was then inserted into pRPA-RD-195, which was previously digested with the restriction enzyme HindIII and dephosphorylated with calf intestinal phosphatase.

pRPA-RD-174: Plasmid derived from pRPA-BL-150A (EP 0 508 909) which contained the bromoxynil tolerance gene from pRPA-BL-237 (EP 0 508 909).

The bromoxynil tolerance gene was isolated from pRPA-BL-237 by PCR gene amplification. The resulting fragment has blunt ends and was cloned in the EcoRI position from pRPA-BL-150A, which was blunted by Klenow polymerase under standard conditions. An Agrobacterium tumefaciens vector was obtained that contained a bromoxynil tolerance gene near its right end, a kanamycin tolerance gene near its left end, and a multiple clonal position between these two genes.

The schematic structure of pRPA-RD-174 is presented in Figure 4. In this figure, "nos" represents the polyadenylation site of nopaline synthase from Agrobacterium tumefaciens (Bevan et al., 1983), "NOS pro" represents the promoter of nopaline synthase from Agrobacterium tumefaciens, "NPT II" represents the neomycin phosphotransferase gene of transposon Tn5 from E. coli (Rothstein et al., 1981), "35S pro" represents the 35S promoter isolated from cauliflower mosaic virus (Odell et al., 1985), "BRX" represents the nitrilase gene isolated from K. ozaenae (Stalker et al., 1988), “RB” and “LB” represent the right and left ends of the Ti plasmid sequence from Agrobacterium tumefaciens, respectively.

pRPA-RD-184: Addition of a new, unique restriction site in pRPA-RD-174.

Complementary synthetic oligonucleotides Oligo 7 and Oligo 8 shown below were hybridized and their double chain was prepared according to the procedure described for pRPA-RD-183.

[image]

Hybridized double-stranded oligonucleotides (96 base pairs) were purified after separation on agar gel (3% Nusieve, FMC). Plasmid pRPA-RD-174 was digested with the restriction enzyme XmaI and the main DNA fragment was purified. The two obtained DNA fragments were then connected to each other.

A plasmid derived from pRPA-RD-174 was obtained which contained other restriction sites between the tolerance gene for bromoxynil and the selection marker of the kanamycin gene.

The schematic structure of plasmid pRPA-RD-184 is shown in Figure 5, in which the terms “nos”, “NPT II”, “NOS pro”, “35S pro”, “BRX gene”, “RB” and “LB” have the same meaning as in Figure 4.

pRPA-RD-236: Preparation of an Agrobacterium tumefaciens vector containing a gene construct encoding androctonin targeting the extracellular matrix.

Plasmid pRPA-RD-233 was digested with restriction enzymes PmeI and AscI, and the DNA fragment containing the PR-1a-androctonin gene was purified. Plasmid pRPA-RD-184 was digested with the same restriction enzymes. The DNA fragment containing the PR-1a-androctonin expression cassette was then inserted into pRPA-RD-184. Thus, a vector was obtained from Agrobacterium tumefaciens that contained a sequence coded for the fusion protein PR-1a-androctonin, which led to the expression of androctonin in the extracellular plant matrix.

Example 2

Tolerance to herbicides in transformed tobacco plants

2.1 - Transformation

The pRPA-RD-236 vector was introduced into the Agrobacterium tumefaciens EHA101 strain (Hood et al., 1987) carrying the pTVK291 cosmid (Komari et al., 1986). The transformation technique is based on the procedure described by Horsh et al. (1985).

2.2 - Regeneration

Regeneration of tobacco plants PBD6 (origin SEITA France) starting from foliar explants was carried out in basic Murashige-Skoog (MS) medium including 30 g/l sucrose and 200 μg/ml kanamycin. Foliar explants were taken in advance from cultivated plants in the greenhouse or in vitro and regenerated by the foliar disk technique (Horsh et al., 1985) in three successive steps: the first step involved the induction of seedlings in a medium supplemented with 30 g/l sucrose containing 0.05 mg/l naphthylacetic acid (NAA) and 2 mg/l benzylaminopurine (BAP) for 2 weeks. The saplings formed during this step were then grown for 10 days by culturing in MS medium to which 30 g/l of sucrose was added but without hormones. Then, the developed saplings were taken out and cultured in MS medium for rooting with half the content of salt, vitamins and sugar, and without the content of hormones. After approximately 2 weeks, the rooted saplings were moved to the greenhouse.

2.3 - Tolerance to bromoxynil

Twenty transformed plants were regenerated and placed in the greenhouse, with the aim of constructing pRPA-RD-236. These plants were treated in the greenhouse with an aqueous suspension according to Pardner, which corresponds to 0.2 kg of bromoxynil active substance per hectare until the stage of 5 leaves.

All plants that showed complete tolerance to bromoxynil were then used for various experiments showing that expression of androctonin in transformed plants results in their resistance to fungal attack.

LITERARY SOURCES

Ausubel, F.A. et al. (eds. Greene), Current Protocols in Molecular Biology, Publ. Wiley & Sons.

Bevan, M. et al., Nuc. Acids Res. 11 (1983) 369-385.

Carrington and Freed, J. Virol. 64 (1990) 1590-1597.

Ehret-Sabatier et al., The Journal of Biological Chemistry, 271, 47 (1996) 29537-29544.

Horsch et al., Science 227 (1985) 1229-1231.

Jefferson et al., EMBO J. 6 (1987) 3901-3907.

Komari et al., J. Bacteriol. 166 (1986) 88-94.

Rothstein et al., Cold Spring Harb. Symp. Quant. Biol. 45 (1981) 99-105.

Stalker et al., J. Biol. Chem. 263 (1988) 6310-6314.

Odell, J.T. et al., Nature 313 (1985) 810-812.

LIST OF SEQUENCES

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: RHONE-POULENC AGROCHIMIE

(B) STREET: 14/20 Rue Pierre BAIZET

(C) CITY: Lyon

(E) COUNTRY: France

(F) POSTAL CODE: 69009

(ii) TITLE OF THE INVENTION: The gene coded for androctonin, the vector containing it, and the resulting disease-resistant transformed plants

(iii) NUMBER OF SEQUENCES: 11

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(D) LOGICAL SUPPORT: PatentIn Release #1.0, Version #1.30 (OEB)

(2) INFORMATION FOR SEQ ID NO 1:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 110 base pairs

(B) TYPE: nucleotide

(C) CHAINING: double

(D) TOPOLOGY: linear

(ii) TYPE OF MOLECULE: DNA

(ix) ADDITIONAL FEATURES:

(A) NAME/KEY: CDS

(B) POSITION: 1..75

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 1:

[image]

(2) INFORMATION FOR SEQ ID NO 2:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 106 base pairs

(B) TYPE: nucleotide

(C) CHAINING: double

(D) TOPOLOGY: linear

(ii) TYPE OF MOLECULE: DNA

(ix) ADDITIONAL FEATURES:

(A) NAME/KEY: CDS

(B) POSITION: 12..101

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 2:

[image]

(2) INFORMATION FOR SEQ ID NO. 3:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 211 base pairs

(B) TYPE: nucleotide

(C) CHAINING: double

(D) TOPOLOGY: linear

(ii) TYPE OF MOLECULE: DNA

(ix) ADDITIONAL FEATURES:

(A) NAME/KEY: CDS

(B) POSITION: 12..176

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 3:

[image]

(2) INFORMATION FOR SEQ ID NO. 4:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 75 base pairs

(B) TYPE: nucleotide

(C) CHAINING: single

(D) TOPOLOGY: linear

(ii) TYPE OF MOLECULE: other nucleic acid

(A) DESCRIPTION: /opis = “synthetic oligonucleotide 1”

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 4:

[image]

(2) INFORMATION FOR SEQ ID NO. 5:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 72 base pairs

(B) TYPE: nucleotide

(C) CHAINING: single

(D) TOPOLOGY: linear

(ii) TYPE OF MOLECULE: other nucleic acid

(A) DESCRIPTION: /opis = “synthetic oligonucleotide 2”

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 5:

[image]

(2) INFORMATION FOR SEQ ID NO. 6:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 44 base pairs

(B) TYPE: nucleotide

(C) CHAINING: single

(D) TOPOLOGY: linear

(ii) TYPE OF MOLECULE: other nucleic acid

(A) DESCRIPTION: /opis = “synthetic oligonucleotide 3”

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 6:

[image]

(2) INFORMATION FOR SEQ ID NO. 7:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 97 base pairs

(B) TYPE: nucleotide

(C) CHAINING: single

(D) TOPOLOGY: linear

(ii) TYPE OF MOLECULE: other nucleic acid

(A) DESCRIPTION: /opis = “synthetic oligonucleotide 4”

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 7:

[image]

(2) INFORMATION FOR SEQ ID NO. 8:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 85 base pairs

(B) TYPE: nucleotide

(C) CHAINING: single

(D) TOPOLOGY: linear

(ii) TYPE OF MOLECULE: other nucleic acid

(A) DESCRIPTION: /opis = “synthetic oligonucleotide 5”

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 8:

[image]

(2) INFORMATION FOR SEQ ID NO. 9:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 66 base pairs

(B) TYPE: nucleotide

(C) CHAINING: single

(D) TOPOLOGY: linear

(ii) TYPE OF MOLECULE: other nucleic acid

(A) DESCRIPTION: /opis = “synthetic oligonucleotide 6”

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 9:

[image]

(2) INFORMATION FOR SEQ ID NO. 10:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 93 base pairs

(B) TYPE: nucleotide

(C) CHAINING: single

(D) TOPOLOGY: linear

(ii) TYPE OF MOLECULE: other nucleic acid

(A) DESCRIPTION: /opis = “synthetic oligonucleotide 7”

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 10:

[image]

(2) INFORMATION FOR SEQ ID NO. 11:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 93 base pairs

(B) TYPE: nucleotide

(C) CHAINING: single

(D) TOPOLOGY: linear

(ii) TYPE OF MOLECULE: other nucleic acid

(A) DESCRIPTION: /opis = “synthetic oligonucleotide 8”

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 11:

[image]

Claims (39)

1. Nucleic acid fragment, characterized in that it contains a nucleic acid sequence coded for androctonin. 2. Nucleic acid fragment according to claim 1, characterized in that it is a DNA sequence. 3. Nucleic acid fragment according to any one of claims 1 or 2, characterized in that androctonin consists of peptides that can be produced by scorpions or can be isolated from them, preferably from the species Androctonus australis, wherein these peptides contain at least 20 amino acids, preferably at least 25 amino acids, and 4 cysteine residues that form disulfide bridges between themselves. 4. Nucleic acid fragment according to any one of claims 1 to 3, characterized in that androctonin essentially consists of a peptide sequence of the general formula (I) Xaa-Cys-Xab-Cys-Xac-Cys-Xad-Cys-Xae (I) where Xaa represents a peptide residue containing at least 1 amino acid, Xab represents a peptide residue of 5 amino acids, Xac represents a peptide residue of 5 amino acids, Xad represents a peptide residue of 3 amino acids, a Xae represents a peptide residue containing at least 1 amino acid. 5. Nucleic acid fragment according to claim 4, characterized in that Xab and/or Xad and/or Xae contain at least one basic amino acid. 6. Nucleic acid fragment according to claim 5, characterized in that the basic amino acids are selected from lysine, asparagine and homoasparagine. 7. Nucleic acid fragment according to any one of claims 4 to 6, characterized in that Xaa represents the peptide sequence Xaa'-Val, in which Xaa' represents NH2 or a peptide residue containing at least 1 amino acid, and/or Xab represents the peptide sequence -Arg-Xab'-Ile, in which Xab' represents a peptide residue of 3 amino acids, and/or Xac represents the peptide sequence -Arg-Xac'-Gly-, in which Xac' represents a peptide residue of 3 amino acids, and/or Xad represents the peptide sequence -Tyr-Xad'-Lys, in which Xad' represents a peptide residue of 1 amino acid, and/or Xae represents the peptide sequence -Thr-Xae', in which Xae' represents COOH or a peptide residue containing at least 1 amino acid. 8. Nucleic acid fragment according to claim 7, characterized in that Xaa' represents the peptide sequence Arg-Ser-, and/or Xab' represents the peptide sequence -Gln-Ile-Lys-, and/or Xac' represents the peptide sequence -Arg-Arg-Gly-, and/or Xad' represents the peptide residue -Tyr-, and/or Xae' represents the peptide sequence -Asn-Arg-Pro-Tyr. 9. Nucleic acid fragment according to one of claims 1 to 8, characterized in that androctonin is represented by a peptide sequence of 25 amino acids described by sequence identifier no. 1 (SEQ ID NO. 1) and homologous peptide sequences. 10. Nucleic acid fragment according to claim 9, characterized in that it is represented by sequence identifier no. 1 (SEQ ID NO. 1), a homologous sequence or a sequence complementary to said sequence, and especially the encrypted part of that SEQ ID NO. 1 corresponding to bases 1 to 75. 11. Nucleic acid fragment, characterized in that it contains a nucleic acid sequence coded for the fusion peptide "peptide-androctonin" or "androctonin-peptide", preferably "peptide-androctonin", wherein androctonin is defined according to one of claims 1 to 9. 12. Nucleic acid fragment according to claim 11, characterized in that the peptide fused with androctonin is a signal peptide or a transit peptide. 13. Nucleic acid fragment according to claim 12, characterized in that the transit peptide is a chloroplast addressing signal or a mitochondrial addressing signal. 14. Nucleic acid fragment according to claim 12, characterized in that the signal peptide is an N-terminal signal or "prepeptide", optionally in combination with a signal responsible for protein retention in the endoplasmic reticulum, or a vacuolar addressing peptide or "propeptide". 15. Nucleic acid fragment according to claim 14, characterized in that the signal peptide represents the signal peptide of the tobacco PR-1α gene. 16. Nucleic acid fragment according to claim 15, characterized in that the fusion peptide "peptide-androctonin" is represented by sequence identifier no. 3 (SEQ ID NO. 3). 17. Nucleic acid fragment according to claim 16, characterized in that it is represented by sequence identifier no. 3 (SEQ ID NO. 3), a homologous sequence or a complementary sequence, and especially the encrypted part of that SEQ ID NO. 3 corresponding to bases 12 to 176 of that sequence. 18. Fusion peptide "peptide-androctonin" or "androctonin-peptide", preferably "peptide-androctonin", characterized in that it is defined according to one of claims 11 to 16. 19. A chimeric gene containing a coded sequence and heterologous regulatory elements in the 5' and 3' positions that can function in the host organism, preferably plant cells or plants, wherein these elements are functionally connected to said coded sequence, indicated by the fact that said coded the sequence contains at least one DNA fragment coding for androctonin, defined according to claims 1 to 17. 20. Chimeric gene according to claim 19, characterized in that the host organism is selected from bacteria, for example E. coli, yeasts, preferably yeasts of the genus Saccharomyces or Kluyveromyces, Pichia, fungi, preferably Aspergillus, baculoviruses, and plant cells and plants. 21. A chimeric gene according to any one of claims 19 and 20, characterized in that it is combined with a selection marker adapted to the transformed host organism. 22. Clonal or expression vector for transformation of the host organism, characterized in that it contains at least one chimeric gene defined according to claims 19 to 21. 23. Process of transformation of host organisms, primarily plant cells, characterized in that it takes place by incorporating at least one nucleic acid fragment or one chimeric gene as defined in claims 19 to 21. 24. The method according to claim 23, characterized in that the chimeric gene is incorporated using the vector according to claim 22. 25. The method according to any one of claims 23 or 24, characterized in that the host organism is selected from among bacteria, for example E. coli, yeasts, preferably yeasts of the genus Saccharomyces or Kluyveromyces, Pichia, fungi, preferably Aspergillus, baculoviruses, and plant cells and plants. 26. The method according to claim 25, characterized in that the host organism is a plant cell. 27. The method according to claim 26, characterized in that the plant is regenerated from transformed plant cells. 28. Transformed host organism, preferably a plant cell or a plant, characterized in that it contains a chimeric gene defined according to one of claims 19 to 21. 29. The host organism according to claim 28, characterized in that it is selected from bacteria, for example E. coli, yeasts, preferably yeasts of the genus Saccharomyces or Kluyveromyces, Pichia, fungi, preferably Aspergillus, baculoviruses, and plant cells and plants. 30. Plants, characterized in that they contain transformed plant cells according to claim 29. 31. The plant according to claim 30, characterized in that it is regenerated from transformed plant cells. 32. A plant, characterized in that it is obtained by growing and/or crossing regenerated plants according to claim 31. 33. The plant according to one of claims 30 to 32, characterized in that it is selected from corn, grain, rapeseed, soybean, rice, sugar cane, sugar beet, tobacco and cotton. 34. The plant according to one of claims 30 to 33, characterized in that it is resistant to fungal diseases such as those caused by Cerospora species, preferably Cerospora beticola, Cladosporium, preferably Cladosporium herbarum, Fusarium, preferably Fusarium culmorum or Fusarium graminearum, or Phytophthora, primarily Phytophthora cinnamoni. 35. Plant seed, indicated by the fact that according to one of the requirements 30 to 34. 36. The process of growing transformed plants according to one of claims 30 to 34, or obtained by the process according to claim 27, characterized in that it consists of sowing the seeds of said transformed plants in the surface of the growing environment, primarily a field suitable for growing said plants, from the application of an agrochemical preparation on said surface, without substantial impact on said transformed seeds or plants, then from harvesting grown plants that have reached the desired level of maturity, and optionally from separating seeds from harvested plants. 37. Cultivation method according to claim 36, characterized in that the agrochemical preparation contains at least one active product that has at least fungicidal and/or bactericidal activity. 38. The method according to claim 37, characterized in that the active product has an activity complementary to that of androctonin produced in transformed plants. 39. A process for the preparation of androctonin defined according to any of claims 1 to 18, characterized in that it includes the steps of growing a transformed host organism defined according to any of claims 28 or 29 in a suitable culture environment, then extraction, and complete or partial purification of the obtained androctonin.