Novel genes encoding insecticidal proteins

Abstract

The present invention relates to novel gene sequences encoding insecticidal proteins produced by Bacillus thuringiensis strains. Particularly, new chimeric genes encoding a Cry1C, Cry1B or Cry1D protein are provided which are useful to protect plants from insect damage. Also included herein are plant cells or plants comprising such genes and methods of making or using them, as well as plant cells or plants comprising one of such chimeric gene and at least one other of such chimeric genes.

1 . A chimeric gene, comprising the following operably-linked sequences:
 a) a coding region encoding a Cry1C protein, comprising the DNA of any one of SEQ ID Nos. 1, 3, 4 or 6, and   b) a promoter region capable of directing expression in plant cells.   
     
     
         2 . The chimeric gene of  claim 1 , wherein said promoter comprises the sequence of SEQ ID No. 18 or 19. 
     
     
         3 . The chimeric gene of  claim 1 , further comprising a 3′ polyadenylation and transcript termination region. 
     
     
         4 . The chimeric gene of  claim 3 , wherein said 3′ polyadenylation and transcript termination region is of the NADP-malic enzyme gene from  Flaveria bidentis.    
     
     
         5 . The chimeric gene of  claim 1 , further comprising the leader sequence of the tapetum specific E1 gene of  Oryza sativa  between the promoter and the coding region. 
     
     
         6 . A DNA comprising the chimeric gene of  claim 1 , further comprising a second chimeric gene, comprising the following operably-linked sequences:
 a) a second coding region encoding a Cry1B protein comprising the DNA of SEQ ID No. 8 or 10, and   b) a second promoter region capable of directing expression in plant cells.   
     
     
         7 . A DNA comprising the chimeric gene of  claim 1 , further comprising a second chimeric gene comprising the following operably-linked sequences:
 a) a coding region encoding a Cry1D protein comprising the DNA of SEQ ID No. 12 or 14, and   b) a promoter region capable of directing expression in plant cells.   
     
     
         8 . The DNA of  claim 6 , wherein said second promoter region comprises the sequence of SEQ ID No. 18 or 19 and is different from said first promoter region. 
     
     
         9 . The DNA of  claim 6 , wherein said second chimeric gene further comprises a 3′ polyadenylation and transcript termination region. 
     
     
         10 . The DNA of  claim 9 , wherein said 3′ polyadenylation and transcript termination region is of the NADP-malic enzyme gene from  Flaveria bidentis.    
     
     
         11 . The DNA of  claim 6 , wherein said second chimeric gene further comprises the leader sequence of the tapetum specific E1 gene of  Oryza sativa  between the promoter and the coding region. 
     
     
         12 . The DNA of  claim 6 , further comprising a third chimeric gene, comprising the following operably-linked sequences:
 a) a coding region encoding a Cry1D protein comprising the DNA of SEQ ID No. 12 or 14, and   b) a promoter region capable of directing expression in plants.   
     
     
         13 . A transgenic plant cell, comprising the gene of  claim 1  stably incorporated in its genome. 
     
     
         14 . A plant comprising the gene of  claim 1  stably incorporated in its genome. 
     
     
         15 . The plant of  claim 14 , which is a  Brassica  species plant or plant cell. 
     
     
         16 . The plant of  claim 15 , wherein said plant is of the species  Brassica oleraceae, Brassica napus, Brassica rapa, Brassica juncea  or  Brassica carinata.    
     
     
         17 . The plant of  claim 16 , wherein said species is cabbage or cauliflower. 
     
     
         18 . (canceled) 
     
     
         19 . A method for controlling insects, comprising: planting or sowing in a field, plants comprising the chimeric gene of  claim 1 . 
     
     
         20 . A chimeric gene comprising the following operably-linked sequences:
 a) a first fragment of a coding sequence encoding an insecticidal protein,   b) a plant intron sequence,   c) a second fragment of said coding sequence,   d) a promoter region capable of directing expression in plant cells.   
     
     
         21 . A method of controlling insects in  Brassica  species plants, comprising: expressing the chimeric genes  claim 1  in plants. 
     
     
         22 . A method of producing plants or seeds resistant to insects, comprising the steps of:
 a) obtaining a plant transformed with the gene of  claim 1 , and   b) selecting progeny of said plant or seeds thereof, containing said gene.   
     
     
         23 . A microorganism comprising the chimeric gene of  claim 1 . 
     
     
         24 . The microorganism of  claim 23 , wherein said microorganism is of the genus  Escherichia, Bacillus,  or  Agrobacterium.    
     
     
         25 . (canceled) 
     
     
         26 . (canceled) 
     
     
         27 . (canceled) 
     
     
         28 . The chimeric gene of  claim 1 , wherein the Cry1C protein is a variant comprising the sequence of SEQ ID No. 2 from amino acid position 29 to amino acid position 627, but wherein one, some or all of the following amino acids at the following positions compared to the positions in SEQ ID No. 2 are changed: the amino acid at position 125 is Alanine, the amino acid at position 184 is Valine, the amino acid at position 295 is Arginine, the amino acid at position 454 is Aspartic acid, or the amino acid at position 593 is Arginine. 
     
     
         29 . The DNA of  claim 6 , wherein the Cry1B protein is a variant comprising the sequence of SEQ ID No. 11 from amino acid position 31 to 648, but wherein the amino acid at position 151 in SEQ ID No.11 is Tyrosine or the amino acid at position 353 in SEQ ID No. 11 is Arginine, or a protein wherein the amino acid at position 151 in SEQ ID No.11 is Tyrosine and the amino acid at position 353 in SEQ ID No. 11 is Arginine. 
     
     
         30 . (canceled) 
     
     
         31 . A transgenic plant cell, comprising the gene of  claim 28  stably incorporated in its genome. 
     
     
         32 . A DNA encoding an optimized chloroplast transit peptide, comprising the sequence of SEQ ID No. 16 from nucleotide position 7 to nucleotide position 371. 
     
     
         33 . The chimeric gene of  claim 20 , wherein the plant intron is the second intron of the ST-LS1 gene of  Solanum tuberosum.    
     
     
         34 . A transgenic plant cell comprising the DNA of  claim 6  stably incorporated in its genome. 
     
     
         35 . A plant comprising the DNA of  claim 6  stably incorporated in its genome. 
     
     
         36 . The plant of  claim 35 , wherein said plant is of the species  Brassica oleraceae, Brassica napus, Brassica rape, Brassica juncea,  or  Brassica carinata.    
     
     
         37 . The plant of  claim 36 , wherein said plant is cabbage or cauliflower. 
     
     
         38 . A method for controlling insects comprising planting or sowing in a field, plants comprising the DNA of  claim 6 . 
     
     
         39 . A method of controlling insects in  Brassica  species plants, comprising expressing the DNA of  claim 6  in said plants. 
     
     
         40 . A method of producing plants or seeds resistant to insects, comprising the steps of:
 a) obtaining a plant transformed with the DNA of  claim 6 , and   b) selecting progeny of said plant or seeds thereof, containing said DNA.   
     
     
         41 . A microorganism comprising the DNA of  claim 6 . 
     
     
         42 . The microorganism of  claim 41 , wherein said microorganism is of the genus  Escherichia, Bacillus,  or  Agrobacterium.