Bacillus Thuringiensis CryET33 and CryET34 Compositions and Uses Therefor

Abstract

Disclosed are Bacillus thuringiensis strains comprising novel crystal proteins which exhibit insecticidal activity against coleopteran insects including red flour beetle larvae ( Tribolium castaneum ) and Japanese beetle larvae ( Popillia japonica ). Also disclosed are novel B. thuringiensis crystal toxin genes, designated cryET33 and cryET34, which encode the colepteran-toxic crystal proteins, CryET33 (29-kDa) crystal protein, and the cryET34 gene encodes the 14-kDa CryET34 crystal protein. The CryET33 and CryET34 crystal proteins are toxic to red flour beetle larvae and Japanese beetle larvae. Also disclosed are methods of making and using transgenic cells comprising the novel nucleic acid sequences of the invention.

1 - 68 . (canceled) 
     
     
         69 . A polynucleotide encoding a crystal protein or peptide with at least 80% amino acid sequence identity to SEQ ID NO:3 and SEQ ID NO:4. 
     
     
         70 . The polynucleotide of  claim 69 , wherein said polynucleotide comprises SEQ ID NO:1 and SEQ ID NO:2. 
     
     
         71 . The polynucleotide of  claim 70 , wherein said polynucleotide comprises SEQ ID NO:11. 
     
     
         72 . The polynucleotide of  claim 69 , wherein said polynucleotide comprises a nucleic acid sequence that hybridizes under stringent conditions to the complement of a nucleic acid sequence comprising SEQ ID NO:1 and SEQ ID NO:2. 
     
     
         73 . The polynucleotide of  claim 69 , wherein said polynucleotide further defined as an RNA segment. 
     
     
         74 . The polynucleotide of  claim 69 , wherein said polynucleotide is operatively linked to a promoter, said promoter expressing said polynucleotide. 
     
     
         75 . A recombinant vector comprising the polynucleotide of  claim 69 . 
     
     
         76 . The recombinant vector of  claim 75 , wherein said recombinant vector is a plasmid, cosmid, phagemid, phage, or virus. 
     
     
         77 . The recombinant vector of  claim 76 , wherein said recombinant vector is pEG246 or pEG1246. 
     
     
         78 . A recombinant host cell comprising the polynucleotide of  claim 69 . 
     
     
         79 . The recombinant host cell of  claim 78 , further defined as a prokaryotic cell. 
     
     
         80 . The recombinant host cell of  claim 79 , further defined as a bacterial cell. 
     
     
         81 . The recombinant host cell of  claim 80 , wherein said bacterial cell is an  E. coli, B. thuringiensis, B. subtilis, B. megaterium , or a  Pseudomonas  sp. cell. 
     
     
         82 . The recombinant host cell of  claim 81 , wherein said bacterial cell is  E. coli  NRRL B-21364,  B. thuringiensis  NRRL B-21366, or  B. thuringiensis  NRRL B-21367. 
     
     
         83 . The recombinant host cell of  claim 78 , defined further as being a eukaryotic cell. 
     
     
         84 . The recombinant host cell of  claim 83 , further defined as a plant cell. 
     
     
         85 . The recombinant host cell of  claim 84 , wherein said plant cell is a corn, wheat, turf grass, vegetable, fruit tree, or ornamental plant cell. 
     
     
         86 . The recombinant host cell of  claim 78 , wherein said host cell expresses said polynucleotide to produce said crystal protein or peptide. 
     
     
         87 . A method of using the polynucleotide of  claim 69  comprising:
 (a) preparing a recombinant vector in which said polynucleotide is positioned under the control of a promoter;   (b) introducing said recombinant vector into a host cell;   (c) culturing said host cell under conditions effective to allow expression of said polynucleotide; and   (d) collecting crystal protein or peptide produced from the expression of said polynucleotide.   
     
     
         88 . The method of  claim 87 , wherein said recombinant vector is pEG246 or pEG1246. 
     
     
         89 . A method of preparing a crystal protein or peptide comprising:
 (a) preparing a recombinant vector comprising the polynucleotide of  claim 69 , wherein said polynucleotide is positioned under the control of a promoter;   (b) introducing said recombinant vector into a host cell;   (c) culturing said host cell under conditions effective to express said polynucleotide; and   (d) collecting crystal protein or peptide produced from the expression of said polynucleotide.   
     
     
         90 . The method of  claim 89 , wherein said recombinant vector is pEG246 or pEG1246. 
     
     
         91 . A method of preparing a crystal protein or peptide comprising:
 (a) culturing the recombinant host cell of  claim 82  under conditions effective to produce a crystal protein or peptide; and   (b) obtaining said crystal protein or peptide from said cell.