Nucleotide sequences coding for polypeptides endowed with a larvicidal activity towards lepidoptera
AssigneeInstitut Pasteur and Institute National de la Recherche Agronomic
InventorsSanchis Vincent, Lereclus Didier, Menou Ghislaine, Lecadet Marguerite-Marie, Martouret Daniel
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Abstract
This invention relates to vectors, bacterial strains, and methods for the cloning and expression of a polypeptide having larvicidal activity. In particular, the invention relates to vectors, bacterial strains and methods for the cloning and expression of the N-terminal region of a polypeptide toxic against the larvae of Lepidoptera of the Noctuidae family, preferably against S. littoralis.
Claims36
1/ Sequence of nucleotides coding for at least a part of the N-terminal region of a polypeptide specifically toxic towards larvae of the Lepidoptera of the family of the Noctuidae, and preferably towards S.littoralis, characterized by its capacity of hybridization with a gene capable of expressing a polypeptide toxic towards larvae of S.littoralis.
2/ Sequence of nucleotides of about 3 kb corresponding to the HindIII-PstI restriction fragment derived from B.thuringiensis capable of hybridizing with the probes 1, 2, 3 of pHTA2 reported in FIG. 2.
3/ Sequence according to claim 1 or 2, characterized in that it contains in the following order the sites:
- HindIII-HincII-Bg1II-KpnI-HindIII-PstI.
4/ Sequence of nucleotides according to any one of the claims 1 to 3, characterized in that it is obtained in vitro from a single strain of B.thuringiensis.
5/ Sequence of nucleotides according to claim 4, characterized in that the strain of B.thuringiensis is the aizawai 7-29 strain.
6/ Sequence of nucleotides according to any one of the claims 1 to 3, characterized in that it is obtained by in vitro genetic recombination of DNA sequences from two different strains of B.thuringiensis.
7/ Sequence according to claim 6, characterized in that the 2 strains of B.thuringiensis correspond to the strains entomocidus 6-01 and aizawai 7-29, respectively.
8/ Sequence of nucleotides, characterized in that it codes for a polypeptide capable of forming an immunological complex with antibodies directed against polypeptides with a larvicidal activity towards S.littoralis.
9/ Sequence of nucleotides characterized in that it has the capacity to hybridize with a probe formed from the sequence (I) exhibiting the following chain arrangement:
- 7 52 GTC TAC TTG ACA GGG GTA GGA ACA TAA TCG GTC AAT TTT AAA TAT GGG GCA TAT ATT GAT 112 ATT TTA TAA AAT TTG TTA CGT TTT TTG TAT TTT TTC ATA AGA TGT GTC ATA TGT ATT AAA 172 TCG TCG TAA TGA AAA ACA GTA TCA AAC TAT CAG AAC TTT GGT AGT TTA ATA AAA AAA CGG 232 ACC TAT TTT ATG GAG GAA AAT AAT CAA AAT CAA TGC ATA CCT TAC AAT TGT TTA AGT AAT 292 CCT GAA GAA GTA CTT TTG GAT GGA GAA CCG ATA TCA ACT GGT AAT TCA TCA ATT GAT ATT 352 TCT CTG TCA CTT CTT CAG TTT CTG GTA TCT AAC TTT GTA CCA GGG GGA GGA TTT TTA GTT 412 GGA TTA ATA GAT TTT GTA TGG CGA ATA CTT CGC CCT TCT CAA TGG GAT CCA TTT CTA GTA 472 CAA ATT GAA CAA TTA ATT AAT GAA AGA ATA GCT GAA TTT GCT AGG AAT GCT GCT ATT GCT 532 AAT TTA GAA GGA TTA GGA AAC AAT TTC AAT ATA TAT GTG GAA GCA TTT AAA GAA TGG CAA 592 GAA GAT CCT AAT AAT CCA GAA ACC AGG ACC AGA GTA ATT GAT CGC TTT CGT ATA CTT GAT 652 CGC CTA CTT GAA AGG GAC ATT CCT TCG TTT CGA ATT TCT GGA TTT GAA GTA CCC CTT TTA 712 TCC GTT TAT GCT CAA GCC CCC AAT CTG CAT CTA GCT ATA TTA AGA GAT TCT GTA ATT TTT 772 GGA GAA AGA TGG GGA TTC ACA ACG ATA AAT GTC AAT GAA AAC TAT AAT AGA CTA ATT AGG 832 CAT ATT GAT GAA TAT GCT GAT GAC TGT CCA AAT ACG TAT AAT CGG GGA TTA AAT AAT TTA 892 CCG AAA TCT ACG TAT CAA GAT TGG ATA ACA TAT AAT CGA TTA CGG AGA GAC TTA ACA TTG 952 ACT GTA TTA GAT ATC GCC GCT TTC TTT CCA AAC TAT GAC
- or from the sequence (III) exhibiting the following chain arrangement:
- 8 1 CAG CTT CAA TAG AAT CTC AAA TCT CGA TGA CTG CTT AGT CTT TTT AAT ACT GTC TAC TTG ACA GGG GTA GAA ACA TAA TCG GTC AAT TTT 91 AAA TAT GGG GCA TAT ATT GAT ATT TTA TAA AGT TTG TTA CGT TTT TTG TAT TTT TTC ATA AGA TGT GTC ATA TGT ATT AAA TCG TGG TAA 181 TGA AAA ACA GTA TCA AAC TAT CAG AAC TTT GGT AGT TTA ATA AAA AAA CGG AGG TAT TTT ATG GAG GAA AAT AAT CAA AAT CAA TGC ATA 271 CCT TAC AAT TGT TTA AGT AAT CCT GAA GAA GTA CTT TTG GAT GGA GAA CGG ATA TCA ACT GGT AAT TCA TCA ATT GAT ATT TCT CTG TCA 361 CTT GTT CAG TTT CTG GTA TCT AAC TTT GTA CCA GGG GGA GGA TTT TTA GTT GGA TTA ATA GAT TTT GTA TGG GGA ATA GTT GGC CCT TCA 451 CAA TGG GAT GCA TTT CTA GTA CAA ATT GAA CAA TTA ATT AAT GAA AGA ATA GCT GAA TTT GCT AGG AAT GCT GCT ATT GCT AAT TTA GAA 541 GGA TTA GGA AAC AAT TTC AAT ATA TAT GTG GAA GCA TTT AAA GAA TGG GAA GAA GAT CCT AAT AAT CCA GCA ACC AGG ACC AGA GTA ATT 631 GAT CGC TTT CGT ATA CTT GAT GGG CTA CTT GAA AGG GAC ATT CCT GCT TTT CGA ATT TCT GGA TTT GAA GTA CCC CTT TTA TCC GTT TAT 721 GCT CAA GCG GCC AAT CTG CAT CTA GCT ATA TTA AGA GAT TCT GTA ATT TTT GGA GAA AGA TGG GGA TTG ACA ACG ATA AAT GTC AAT GAA 811 AAC TAT AAT AGA CTA ATT AGG CAT ATT GAT GAA TAT GCT GAT CAC TGT GCA AAT ACG TAT AAT CGG GGA TTA AAT AAT TTA CCG AAA TCT 901 ACG TAT CAA GAT TGG ATA ACA TAT AAT CGA TTA CGG AGA GAC TTA ACA TTG ACT GTA TTA GAT ATC GCC GCT TTC TTT CCA AAC TAT GAC 991 AAT AGG AGA TAT CCA ATT CAG CCA GTT GGT CAA CTA ACA AGG GAA GTT TAT ACG GAC CCA TTA ATT GAT TTT AAT CCA CAG TTA CAG TCT 1081 GTA GCT CAA TTA CCT ACT TTT AAC GTT ATG GAG AGC AGC GCA ATT AGA AAT CCT CAT TTA TTT GAT ATA TTG AAT AAT CTT ACA ATC TTT 1171 ACG GAT TGG TTT AGT GTT GGA CGC AAT TTT TAT TGG GGA GGA CAT CGA GTA ATA TCT AGC CTT ATA GGA GGT GGT AAC ATA ACA TCT CCT 1261 ATA TAT GGA AGA GAG GCG AAC CAG GAG CCT CCA AGA TCC TTT ACT TTT AAT GGA CCG GTA TTT AGG ACT TTA TCA ATT CCT ACT TTA CGA 1351 TTA TTA CAG CAA CCT TGC CAG CGC CAC CAT TTT AAT TTA CGT GGT GGT GAA GGA GTA GAA TTT TCT ACA CCT ACA AAT AGC TTT ACG TAT 1441 CGA GGA AGA GGT ACG GTT GAT TCT TTA ACT GAA TTA CCG CCT GAG GAT AAT AGT GTG CCA CCT CGC GAA GGA TAT AGT CAT CGT TTA TGT 1521 CAT GCA ACT TTT GTT CAA AGA TCT GGA ACA CCT TTT TTA ACA ACT GGT GTA GTA TTT TCT TGG ACG CAT CGT AGT GCA ACT CTT ACA AAT 1621 ACA ATT GAT CCA GAG AGA ATT AAT CAA ATA CCT TTA GTG AAA GGA TTT AGA GTT TGG GGG GGC ACC TCT GTC ATT ACA GGA CCA GGA TTT 1711 ACA GGA GGG GAT ATC CTT CGA AGA AAT ACC TTT GGT GAT TTT GTA TCT CTA CAA GTC AAT ATT AAT TCA CCA ATT ACC CAA AGA TAC CGT 1801 TTA AGA TTT CGT TAC GCT TCC AGT AGG GAT GCA CGA GTT ATA GTA TTA ACA GGA GCG GCA TCC ACA GGA GTG GGA GGC CAA GTT AGT GTA 1891 GAT ATG CCT CTT CAG AAA ACT ATG GAA ATA GGG TAG AAC TTA ACA TCT AGA ACA TTT AGA TAT ACC GAT TTT AGT AAT CCT TTT TCA TTT 1901 AGA GCT AAT CCA GAT ATA ATT GGG ATA AGT GAA CAA CCT CTA TTT GGT GCA GGT TCT ATT AGT AGC GGT GAA CTT TAT ATA GAT AAA ATT 2071 GAG ATT ATT CTA GCA GAT GCA ACA TTT GAA GCA GAA TCT GAT TTA GAA AGA GCA CAA AAG GCG GTG AAT GCC CTG TTT ACT TCT TCC AAT 2161 CAA ATC GGG TTA AAA ACC GAT GTG ACG GAT TAT CAT ATT GAT CAA GTA TCC AAT TTA GTG GAT TGT TTA TCA GAT GAA TTT TGT CTG GAT 2251 GAA AAG CGA GAA TTG TCC GAG AAA GTC AAA CAT GCG AAG CGA CTC AGT GAT GAG CGG AAT TTA CTT CAA GAT CCA AAC TTC AGA GGG ATC 2341 GAT AGA CAA CCA GAC CGT GGC TGG AGA GGA AGT ACA GAT ATT ACC ATC CAA GCA GGA GAT GAC GTA TTC AAA GAG AAT TAC GTC ACA CTA 2431 CCG GGT ACC GTT GAT GAG TGC TAT CCA ACG TAT TTA TAT CAG AAA ATA GAT GAG TCG AAA TTA AAA GCT TAT ACC CGT TAT GAA TTA AGA 2521 GGG TAT ATC GAA GAT AGT CAA GAC TTA GAA ATC TAT TTG ATC GCG TAC AAT GCA AAA CAC GAA ATA GTA AAT GTG CCA GGC ACG GGT TCC 2611 TTA TGG CCG CTT TCA GCC CAA AGT CCA ATC GGA AAG TGT GGA GAA CCG AAT CGA TGC CGC CCA CAC CTT GAA TGG AAT CCT GAT CTA GAT 2701 TGT TCC TGC AG
10/ Sequence of nucleotides coding for a polypeptide toxic specifically towards larvae of the Lepidoptera of the family of the Noctuidae, and preferably towards S.littoralis, characterized in that it includes the chain arrangement (I) or (III) defined in claim 9.
11/ Sequence of nucleotides according to claim 9 or 10, characterized in that it has an ATG initiation codon situated at position 241.
12/ Sequence according to any one of the claims 9 to 11, characterized by a GGAGG binding site to ribosomes at positions 230 to 234.
13/ Sequence according to one of the claims 10 to 12, characterized in that it contains the sequence included between the nucleotides at position 137 and 177 (position −103 to −63) upstream from the ATG initiation codon) which is homologous to the extent of about at least 70% with the region present upstream from the gene for the crystal of the strain kurstaki-HD1 Dipel (BTK) which contains the three promoters BtI, BtII and Ec, functional in B.thuringiensis and E.coli, respectively.
14/ Sequence of nucleotides, characterized in that it codes for a polypeptide comprising the sequence of amino acids (II) below:
- 9 MET GLU GLU ASN ASN GLN ASN GLN CYS ILE PRO TYR ASN CYS LEU SER ASN PRO GLU GLU VAL LEU LEU ASP GLY GLU ARG ILE SER THR GLY ASN SER SER ILE ASP ILE SER LEU SER LEU VAL GLN PHE LEU VAL SER ASN PHE VAL PRO GLY GLY GLY PHE LEU VAL GLY LEU ILE ASP PHE VAL TRP GLY ILE VAL GLY PRO SER GLN TRP ASP ALA PHE LEU VAL GLN ILE GLU GLN LEU ILE ASN GLU ARG ILE ALA GLU PHE ALA ARG ASN ALA ALA ILE ALA ASN LEU GLU GLY LEU GLY ASN ASN PHE ASN ILE TYR VAL GLU ALA PHE LYS GLU TRP GLU GLU ASP PRO ASN ASN PRO GLU THR ARG THR ARG VAL ILE ASP ARG PHE ARG ILE LEU ASP GLY LEU LEU GLU ARG ASP ILE PRO SER PHE ARG ILE SER GLY PHE GLU VAL PRO LEU LEU SER VAL TYR ALA GLN ALA ALA ASN LEU HIS LEU ALA ILE LEU ARG ASP SER VAL ILE PHE GLY GLU ARG TRP GLY LEU THR THR ILE ASN VAL ASN GLU ASN TYR ASN ARG LEU ILE ARG HIS ILE ASP GLU TYR ALA ASP HIS CYS ALA ASN THR TYR ASN ARG GLY LEU ASN ASN LEU PRO LYS SER THR TYR GLN ASP TRP ILE THR TYR ASN ARG LEU ARG ARG ASP LEU THR LEU THR VAL LEU ASP ILE ALA ALA PHE PHE PRO ASN TYR ASP
- or that it codes for a polypeptide comprising the sequence of amino acids (IV) below:
- 10 MET GLU GLU ASN ASN GLN ASN GLN LYS ILE 271 PRO TYR ASN CYS LEU SER ASN PRO GLU GLU VAL LEU LEU ASP GLY GLU ARG ILE SER THR GLY ASN SER SER ILE ASP ILE SER LEU SER 361 LEU VAL GLN PHE LEU VAL SER ASN PHE VAL PRO GLY GLY GLY PHE LEU VAL GLY LEU ILE ASP PHE VAL TRP GLY ILE VAL GLY PRO SER 451 GLN TRP ASP ALA PHE LEU VAL GLN ILE GLU GLN LEU ILE ASN GLU ARG ILE ALA GLU PHE ALA ARG ASN ALA ALA ILE ALA ASN LEU GLU 541 GLY LEU GLY ASN ASN PHL ASN ILE TYR VAL GLU ALA PHE LYS GLU TRP GLU GLU ASP PRO ASN ASN PRO ALA THR ARG THR ARG VAL ILE 631 ASP ARG PHE ARG ILE LEU ASP GLY LEU LEU GLU ARG ASP ILE PRO SER PHE ARG ILE SER GLY PHE GLU VAL PRO LEU LEU SER VAL TYR 721 ALA GLN ALA ALA ASN LEU HIS LEU ALA ILE LEU ARG ASP SER VAL ILE PHE GLY GLU ARG TRP GLY LEU THR THR ILE ASN VAL ASN GLU 811 ASA TYR ASN ARG LEU ILE ARG HIS ILE ASP GLU TYR ALA ASP HIS CYS ALA ASN THR TYR ASN ARG GLY LEU ASN ASN LEU PRO LYS SER 901 THR TYR GLN ASP TRP ILE THR TYR ASN ARG LEU ARG ARG ASP LEU THR LEU THR VAL LEU ASP ILE ALA ALA PHE PHE PRO ASN TYR ASP 991 ASN ARG ARG TYR PRO ILE GLN PRO VAL GLY GLN LEU THR ARG GLU VAL TYR THR ASP PRO LEU ILE ASN PHE ASN PRO GLN LEU GLN SER 1061 VAL ALA GLN LEU PRO THR PHE ASN VAL MET GLU SER SER ALA ILE ARG ASN PRO HIS LEU PHE ASP ILE LEU ASN ASN LEU THR ILE PHE 1171 THR ASP TRP PHE SER VAL GLY ARG ASN PHE TYR TRP GLY GLY HIS ARG VAL ILE SER SER LEU ILE GLY GLY GLY ASN ILE THR SER PRO 1261 ILE TYR GLY ARG GLU ALA ASN GLN GLU PRO PRO ARG SER PHE THR PHE ASN GLY PRO VAL PHE ARG THR LEU SER ILE PRO THR LEU ARG 1351 LEU LEU GLN GLN PRO CYS GLN ARG HIS HIS PHE ASN LEU ARG GLY GLY GLU GLY VAL GLU PHE SER THR PRO THR ASN SER PHE THR TYR 1441 ARG GLY ARG GLY THR VAL ASP SER LEU THR GLU LEU PRO PRO GLU ASP ASN SER VAL PRO PRO ARG GLU GLY TYR SER HIS ARG LEU CYS 1521 HIS ALA THR PHE VAL GLN ARG SER GLY THR PRO PHE LEU THR THR GLY VAL VAL PHE SER TRP THR HIS ARG SER ALA THR LEU THR ASN 1621 THR ILE ASP PRO GLU ARG ILE ASN GLN ILE PRO LEU VAL LYS GLY PHE ARG VAL TRP GLY GLY THR SER VAL ILE THR GLY PRO GLY PHE 1711 THR GLY GLY ASP ILE LEU ARG ARG ASN THR PHE GLY ASP PHE VAL SER LEU GLN VAL ASN ILE ASN SER PRO ILE THR GLN ARG TYR ARG 1801 LEU ARG PHE ARG TYR ALA SER SER ARG ASP ALA ARG VAL ILE VAL LEU THR GLY ALA ALA SER THR GLY VAL GLY GLY GLN VAL SER VAL 1891 ASN MET PRO LEU GLN LYS THR MET GLU ILE GLY GLU ASN LEU THR SER ARG THR PHE ARG TYR THR ASP PHE SER ASN PRO PHE SER PHE 1901 ARG ALA ASN PRO ASP ILE ILE GLY ILE SER GLU GLN PRO LEU PHE GLY ALA GLY SER ILE SER SER GLY GLU LEU TYR ILE ASP LYS ILE 2071 GLU ILE ILE LEU ALA ASP ALA THR PHE GLU ALA GLU SER ASP LEU GLU ARG ALA GLN LYS ALA VAL ASN ALA LEU PHE THR SER SER ASN 2161 GLN ILE GLY LEU LYS THR ASP VAL THR ASP TYR HIS ILE ASP GLN VAL SER ASN LEU VAL ASP CYS LEU SER ASP GLU PHE CYS LEU ASP 2231 GLU LYS ARG GLU LEU SER GLU LYS VAL LYS HIS ALA LYS ARG LEU SER ASP GLU ARG ASN LEU LEU GLN ASP PRO ASN PHE ARG GLY ILE 2341 ASN ARG GLN PRO ASP ARG GLY TRP ARG GLY SER THR ASP ILE THR ILE GLN GLY GLY ASP ASP VAL PHE LYS GLU ASN TYR VAL THR LEU 2431 PRO GLY THR VAL ASP GLU CYS TYR PRO THR TYR LEU TYR GLN LYS ILE ASP GLU SER LYS LEU LYS ALA TYR THR ARG TYR GLU LEU ARG 2521 GLY TYR ILE GLU ASP SER GLN ASP LEU GLU ILE TYR LEU ILE ALA TYR ASN ALA LYS HIS GLU ILE VAL ASN VAL PRO GLY THR GLY SER 2611 LEU TRP PRO LEU SER ALA GLN SER PRO ILE GLY LYS CYS GLY GLU PRO ASN ARG CYS ALA PRO HIS LEU GLU TRP ASN PRO ASP LEU ASP 2701 CYS SER CYS
15/ Recombinant expression and cloning vector containing at least a part of the nucleotide sequence such as defined in any one of the claims 1 to 14.
16/ Plasmid according to claim 15 characterized in that it is pHT671 as represented in FIG. 4, or pHT71 comprising a HindIII-PstI DNA fragment constituted uniquely of DNA derived from the aizawai 7-29 strain.
17/ Modified bacterial strains, characterized in that after transformation they contain a sequence of nucleotides according to one of the claims 1 to 14.
18/ Bacterial strain according to claim 17, characterized in that it contains at least one recombinant vector according to claim 15 or 16.
19/ Polypeptide toxic towards larvae of the Lepidoptera and preferentially towards S.littoralis, characterized in that it is capable of forming an immunological complex with antibodies directed against polypeptides with larvicidal activity towards S.littoralis.
20/ Polypeptide according to claim 19, characterized in that it contains the sequence (II) or the sequence (IV) of amino acids defined in claim 14.
21/ Procedure for obtaining a nucleotide sequence coding for at least a part of the N-terminal region of a polypeptide toxic specifically towards larvae of the Lepidoptora of the family of the Noctuidae, and preferentially towards S.littoralis, characterized by the following steps:
- the carrying out of a hybridization between a sequence of nucleotides from a strain of B.thuringiensis active against S.littoralis, on the one hand, and, on the other, one or several sequences of nucleotides utilized as probes derived from the 5′ part of a restriction fragment of a gene for a δ-endotoxin of B.thuringiensis, this part coding for the N-terminal part of a polypeptide toxic towards the Lepidoptera, and derived from the 3′ part of this fragment coding for the COOH part of the polypeptide,
- the isolation of the fragment,
- its cloning in a vector, followed by its purification.
22/ Procedure according to claim 21, characterized in that the hybridization probes utilized are obtained from a gene for a δ-endotoxin derived from a aizawai 7-29 strain coding for a protein of 130 kDa active against P.brassicae and inactive towards S.littoralis, this gene having been cloned in the recombinant plasmid pHTA2.
23/ Procedure according to claim 21 or 22, characterized in that the fragment recombined with the vector in the cloning step is elaborated from at least one sequence of nucleotides derived from at least one recombinant vector containing a sequence of nucleotides from at least one strain of B.thuringiensis.
24/ Procedure according to claim 23, characterized in that the fragment recombined with the vector in the cloning step is elaborated from several sequences of nucleotides derived from recombinant vectors containing sequences of nucleotides from at least 2 different strains of B.thuringiensis, possessing the same restriction maps and themselves containing all or part of the sequences of nucleotides capable of coding for a polypeptide active preferentially towards S.littoralis.
25/ Procedure according to claim 23, characterized in that the fragment recombined with the vector in the cloning step is elaborated from a HindIII-PstI restriction fragment derived from the aizawai 7-29 strain.
26/ Procedure according to claim 24, characterized in that the fragment recombined with the vector in the cloning step is elaborated from a hindIII-HincII restriction fragment derived from the entomocidus 6-01 strain and from a HincII-PstI restriction fragment derived from the aizawai 7-29 strain.
27/ Procedure according to claim 22, characterized in that the restriction fragment recombined according to claim 25 is carried preferentially by a plasmid pHTA6 and the restriction fragments recombined according to claim 26, HindIII-HincII and HincII-PstI, are carried preferentially by the respective recombinant plasmids pHTE6 and pHTA6, the said plasmids pHTA6 and pHTE6 being those isolated with the aid of a probe constituted by a PvuII fragment of 2 kb of the plasmid pBT15-88 corresponding to the internal part of a gene for the chromosomal crystal of the berliner 1715 strain, from transforming clones containing nucleotide sequences derived from B.thuringiensis strains active towards larvae of the Lepidoptera, inter-alia S.littoralis.
28/ Larvicidal composition with preferential activity towards S.littoralis, characterized in that it contains an efficacious amount of polypeptide such as defined in any one of the claims 19 to 20 expressed by the nucleotide sequences according to any one of the claims 1 to 14, the vector according to the claim 15, or the plasmid according to the claim 16, or the bacterial strain according to any one of the claims 17 or 18.
29/ Application of the nucleotide sequences according to any one of the claims 1 to 14 to produce a polypeptide toxic towards Lepidoptera, and preferentially S.littoralis, in microorganisms capable of expressing recombinant vectors containing these sequences such as E.coli, B.subtilis, B.cereus or B.thuringiensis.
30/ Application according to claim 29, characterized in that the sequences of nucleotides are introduced into microorganisms living in the environment or in association with the plants such as Pseudomonas, Azospirillum or Rhizobium and capable of expressing recombinant vectors containing these sequences.
31/ Application according to claim 29 or 30, characterized in that the nucleotide sequences are introduced into microorganisms in combination with different δ-endotoxin genes.
32/ Application of the nucleotide sequences according to any one of the claims 1 to 14 to the transformation of plants sensitive to S.littoralis, characterized in that it comprises the transfer and the expression of these sequences in these plants.
33/ Plant cells, the genome of which, after transformation by means of a non-essentially biological procedure, possesses in a stable manner a sequence of nucleotides capable of expressing a polypeptide toxic towards S.littoralis, such as defined in any one of the claims 1 to 14 and cells derived from their division.
34/ Plants having in particular S.littoralis as predator, transformed by a non-essentially biological procedure, the genome of which possesses in a stable manner a sequence of nucleotides such as defined in any one of the claims 1 to 14, capable of expressing a polypeptide toxic towards S.littoralis and plants derived from their reproduction, their multiplication, or hybrid crosses.
35/ Plant having in particular S.littoralis as predator, possessing in addition to their initial phenotypic and genotypic characters the property of expressing a polypeptide toxic preferentially towards S.littoralis, this property resulting from the insertion in its genome by genetic manipulation of a sequence of nucleotides capable of expressing the said polypeptide.
36/ Seed capable of giving rise to a plant according to claim 34 or 35 or derived from such a plant, characterized in that it has integrated into its genome, by genetic manipulation, a sequence of nucleotides according to any one of the claims 1 to 14.