Chimeric gene encoding drosomycin, vector containing it and production of disease-resistant transgenic plants

Abstract

The invention concerns a chimeric gene containing a DNA sequence coding for drosomycin, a vector containing the chimeric gene, a method for transforming plants and the resulting transformed plants. The drosomycin produced by the plants provides them with resistance to diseases, in particular of fungal origin.

1 . Chimeric gene comprising a coding sequence as well as heterologous regulatory elements at positions 5′ and 3′ capable of functioning in a plant, characterized in that the coding sequence comprises at least one DNA sequence encoding drosomycin.  
     
     
         2 . Chimeric gene according to  claim 1 , characterized in that the drosomycin essentially comprises the peptide sequence of formula (I) below: 
       Xaa-Cys-Xab-Cys-Xac-Cys-Xad-Cys-Xae-Cys-Xaf-Cys-Xag-Cys-Xah-Cys  (I) 
       in which 
 Xaa represents a peptide residue comprising at least 1 amino acid,  
 Xab represents a peptide residue of 8 amino acids,  
 Xac represents a peptide residue of 7 amino acids,  
 Xad represents a peptide residue of 3 amino acids,  
 Xae represents a peptide residue of 9 amino acids,  
 Xaf represents a peptide residue of 5 amino acids,  
 Xag represents a peptide residue of one amino acid, and  
 Xah represents a peptide residue of 2 amino acids.  
 
     
     
         3 . Chimeric gene according to  claim 2 , characterized in that Xab and/or Xad and/or Xac comprise at least one basic amino acid.  
     
     
         4 . Chimeric gene according to  claim 3 , characterized in that Xab comprises at least 2 basic amino acids, preferably 2 and/or Xad and/or Xaf comprise at least 1 basic amino acid, preferably 1.  
     
     
         5 . Chimeric gene according to either of claims  2  and  3 , characterized in that 
 Xaa represents the peptide sequence Xaa′-Asp- in which Xaa′ represents NH 2  or a peptide residue comprising at least 1 amino acid, and/or  
 Xab represents the peptide sequence -Leu-Xab′-Pro- in which Xab′ represents a peptide residue of 6 amino acids, and/or  
 Xac represents the peptide sequence -Ala-Xac′-Thr- in which Xac′ represents a peptide residue of 5 amino acids, and/or  
 Xad represents the peptide sequence -Arg-Xad′-Val, in which Xad′ represents a peptide residue of one amino acid, and/or  
 Xae represents the peptide sequence -Lys-Xae′-His- in which Xae′ represents a peptide residue of 7 amino acids, and/or  
 Xaf represents the peptide sequence -Ser-Xaf′-Lys- in which Xaf′ represents a peptide residue of 3 amino acids, and/or  
 Xag represents Trp, and/or  
 Xah represents the peptide residue Glu-Gly.  
 
     
     
         6 . Chimeric gene according to  claim 5 , characterized in that 
 Xab′ represents the peptide sequence Ser-Gly-Arg-Tyr-Lys-Gly, and/or    Xac′ represents the peptide sequence Val-Trp-Asp-Asn-Glu, and/or    Xad′ represents Arg, and/or    Xae′ represents the peptide sequence Glu-Glu-Gly-Arg-Ser-Ser-Gly, and/or    Xaf′ represents the peptide sequence Pro-Ser-Leu.    
     
     
         7 . Chimeric gene according to one of  claims 1  to  6 , characterized in that the drosomycin is the peptide sequence represented by the sequence identifier No. 4 (SEQ ID No. 4) and the homologous peptide sequences.  
     
     
         8 . Chimeric gene according to one of  claims 1  to  7 , characterized in that the drosomycin is a “peptide-drosomycin” fusion peptide, the cutting of which by the enzymatic systems of plant cells allows the liberation of the drosomycin defined in  claims 1  to  7 .  
     
     
         9 . Chimeric gene according to  claim 8 , characterized in that the “peptide-drosomycin” fusion peptide is represented by the sequence identifier No. 6 (SEQ ID No. 6).  
     
     
         10 . “Peptide-drosomycin” fusion peptide, characterized in that the drosomycin is defined according to  claims 1  to  7 .  
     
     
         11 . Fusion peptide according to  claim 10 , characterized in that the peptide is to be a signal peptide or a transit peptide.  
     
     
         12 . Fusion peptide according to  claim 11 , characterized in that the signal peptide is chosen from the signal peptide of the tobacco PR-1α gene or ubiquitin.  
     
     
         13 . Chimeric gene according to one of  claims 1  to  12 , characterized in that it comprises, in addition, at least one herbicide tolerance gene.  
     
     
         14 . Chimeric gene according to one of  claims 1  to  14 , characterized in that it comprises, in addition, at least one sequence encoding another peptide capable of conferring on the plant resistance to other diseases of bacterial or fungal origin.  
     
     
         15 . Chimeric gene according to one of  claims 1  to  14 , characterized in that the regulatory elements comprise promoter sequences, transcription activators, transit peptides and/or terminator sequences.  
     
     
         16 . Chimeric gene according to one of  claims 1  to  14 , characterized in that the regulatory promoter sequence is chosen from promoter sequences of bacterial, viral or plant origin.  
     
     
         17 . Chimeric gene according to  claim 16 , characterized in that the regulatory promoter sequence is chosen from the promoter of the gene for the ribulose-bisphosphate carboxylase/oxygenase (RuBisCO) small subunit or that of the cauliflower mosaic (CAMV 19S or 35S).  
     
     
         18 . Chimeric gene according to  claim 16 , characterized in that the regulatory promoter sequence comprises at least one promoter chosen from histone or actin promoters.  
     
     
         19 . Vector for transforming plants, characterized in that it contains at least one chimeric gene according to one of  claims 1  to  18 .  
     
     
         20 . Transformed plant cell containing at least one DNA as defined in one of  claims 1  to  18 .  
     
     
         21 . Disease-resistant plant, characterized in that it comprises a transformed cell according to  claim 20 .  
     
     
         22 . Plant according to  claim 21 , characterized in that it is obtained by regeneration from a transformed cell according to  claim 20 .  
     
     
         23 . Disease-resistant transformed plant, characterized in that it is derived from the cultivation and/or the crossing of plants according to either of claims  21  and  22 .  
     
     
         24 . Seeds of transformed plants according to one of  claims 21  to  23 .  
     
     
         25 . Method of transforming plants to make them resistant to diseases, characterized in that a chimeric gene according to one of  claims 1  to  18  is inserted.  
     
     
         26 . Method of transforming plants to make them resistant to fungal diseases, characterized in that a chimeric gene according to one of  claims 1  to  18  is inserted.  
     
     
         27 . Method according to either of claims  25  and  26 , characterized in that the transfer is carried out with  Agrobacterium tumefaciens  or  Agrobacterium rhizogenes.    
     
     
         28 . Method according to either of claims  25  and  26 , characterized in that the transfer is carried out by supplying by bombardment with the aid of particles charged with DNA.  
     
     
         29 . Method according to one of  claims 25  to  28 , characterized in that at least one herbicide tolerance gene is also inserted.  
     
     
         30 . Method according to one of  claims 25  to  29 , characterized in that at least one sequence encoding another peptide capable of conferring on the plant resistance to other diseases of bacterial or fungal origin is also inserted.  
     
     
         31 . Method of cultivating the transformed plants according to one of  claims 21  to  24 , characterized in that it consists in planting the seeds of the said transformed plants in an area of a field appropriate for the cultivation of the said plants, in applying to the said area of the said field an agrochemical composition, without substantially affecting the said seeds or the said transformed plants, and then in harvesting the cultivated plants when they reach the desired maturity and optionally in separating the seeds from the harvested plants.  
     
     
         32 . Method of cultivation according to  claim 31 , characterized in that the agrochemical composition comprises at least one active product having at least one fungicidal and/or bactericidal activity.  
     
     
         33 . Method of cultivation according to  claim 24 , characterized in that the active product exhibits activity complementary to that of drosomycin.