Nucleic acid sequences from diabrotica virgifera virgifera leconte and uses thereof

Abstract

Expressed Sequence Tags (ESTs) isolated from the Western Corn Rootworm, Diabrotica virgifera virgifera LeConte, are disclosed. The invention encompasses nucleic acid molecules that encode D. v. virgifera protein homologs and fragments thereof. In addition, antibodies capable of binding the proteins are encompassed by the present invention. The disclosed ESTs have particular utility in isolating genes and promoters, identifying and mapping the genes involved in developmental and metabolic pathways, and determining gene function. The ESTs provide a unique molecular tool for the targeting and isolation of novel genes for plant protection and improvement. The invention also relates to methods of using the disclosed nucleic acid molecules, proteins, fragments of proteins, and antibodies, for example, for gene identification and analysis, and preparation of constructs.

What is claimed is: 
     
         1 . A substantially purified nucleic acid molecule comprising a nucleotide sequence which is or is complementary to or which is a degenerate variant of a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 through SEQ ID NO: 9112. 
     
     
         2 . A substantially purified nucleic acid molecule according to  claim 1 , wherein said nucleic acid molecule encodes a  D. v. virgifera  protein or fragment thereof. 
     
     
         3 . A substantially purified nucleic acid molecule according to  claim 2 , wherein said  D. v. virgifera  protein or fragment thereof is a receptor or fragment thereof which specifically binds a bacterial protein. 
     
     
         4 . A substantially purified nucleic acid molecule according to  claim 3 , wherein said bacterial protein is an insecticidal protein. 
     
     
         5 . A substantially purified nucleic acid molecule according to  claim 4 , wherein said insecticidal protein is selected from the group consisting of insecticidal proteins produced by  B. thuringiensis, B. sphaericus, Photorhabdus , and  Xenorhabdus  species. 
     
     
         6 . A substantially purified nucleic acid molecule according to  claim 2 , wherein said  D. v. virgifera  protein or fragment thereof is a homologue of a non- D. v. virgifera  insect protein or fragment thereof. 
     
     
         7 . A substantially purified nucleic acid molecule according to  claim 2 , wherein said  D. v. virgifera  protein or fragment thereof is a homologue of a protein or fragment thereof produced by a bacterium or eukaryotic organism, wherein said eukaryotic organism is selected from the group consisting of plant, fungi, algae, arachnid, invertebrate animal, and vertebrate animal. 
     
     
         8 . A substantially purified nucleic acid molecule according to  claim 2 , wherein said  D. v. virgifera  protein or fragments thereof exhibits a percentage identity with a homologue of between about 90% identity and 99% identity. 
     
     
         9 . A substantially purified nucleic acid molecule according to  claim 2 , wherein said  D. v. virgifera  protein or fragments thereof exhibits a percentage identity with a homologue of between about 70% identity and about 90% identity. 
     
     
         10 . A substantially purified first nucleic acid molecule capable of specifically hybridizing to a second nucleic acid molecule which is or is complementary to a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 through SEQ ID NO: 9112. 
     
     
         11 . A transformed cell comprising a polynucleotide sequence which comprises:
 a) a promoter sequence which functions in said cell and is operably linked to   b) a structural nucleic acid comprising a first nucleotide sequence which is or is complementary to a second nucleotide sequence selected from the group consisting of SEQ ID NO: 1 through SEQ ID NO: 9112, wherein said second nucleotide sequence encodes a  D. v. virgifera  protein or fragment thereof and is operably linked to   c) a 3′ non-translated sequence that functions in said cell to cause termination of transcription.   
     
     
         12 . The transformed cell according to  claim 11 , wherein said cell is selected from the group consisting of an insect cell, a mammalian cell, a plant cell, an algal cell, a bacterial cell, and a fungal cell. 
     
     
         13 . The transformed cell according to  claim 11 , wherein said  D. v. virgifera  protein or fragment thereof is a receptor or fragment thereof, wherein said receptor binds a bacterial protein. 
     
     
         14 . The transformed cell according to  claim 12 , wherein said bacterial protein is an insecticidal protein toxin. 
     
     
         15 . The transformed cell according to  claim 14 , wherein said toxin is selected from the group consisting of toxins produced by  B. thuringiensis, B. sphaericus, Photorhabdus , and  Xenorhabdus  species. 
     
     
         16 . The transformed cell according to  claim 15 , wherein said receptor is disposed at the surface of said cell. 
     
     
         17 . A method for screening a candidate toxin comprising:
 a) culturing cells comprising a polynucleotide sequence which comprises: i) a promoter sequence which functions in said cell and is operably linked to ii) a structural nucleic acid comprising a first nucleotide sequence which is or is complementary to a second nucleotide sequence selected from the group consisting of SEQ ID NO: 1 through SEQ ID NO: 9112, wherein said second nucleotide sequence encodes a  D. v. virgifera  protein or fragment thereof which is a receptor or fragment thereof disposed at the surface of said cells, wherein said receptor binds an insecticidal protein toxin selected from the group consisting of toxins produced by  B. thuringiensis, B. sphaericus, Photorhabdus , and  Xenorhabdus  species, linked to iii) a 3′ non-translated sequence that functions in said cell to cause termination of transcription;   b) recovering said cells;   c) contacting said cells with said candidate toxin; and   d) determining effects of said candidate toxin on metabolism or morphology of said cells, wherein said determination is predictive of a cytotoxic or cytostatic property of said candidate toxin.   
     
     
         18 . A transformed plant cell comprising a polynucleotide sequence which comprises:
 a) an exogenous promoter sequence which functions in a plant cell to cause the production of a mRNA molecule; which is operably linked to   b) a structural nucleic acid molecule comprising a first nucleotide sequence which is or is complementary to a second nucleotide sequence selected from the group consisting of SEQ ID NO: 1 through SEQ ID NO: 9112; which is operably linked to   c) a 3′ non-translated sequence that functions in said plant cell to cause termination of transcription and addition of polyadenylated ribonucleotides to the 3′ end of said mRNA molecule.   
     
     
         19 . The transformed cell according to  claim 18 , wherein said cell is selected from the group consisting of a monocot and a dicot plant cell. 
     
     
         20 . A computer readable medium having recorded thereon one or more of the nucleotide sequences as set forth in SEQ ID NO: 1 through SEQ ID NO: 9112 or complements thereof. 
     
     
         21 . A computer readable medium according to  claim 20 , wherein said nucleotide sequences as set forth in SEQ ID NO: 1 through SEQ ID NO: 9112 are recorded thereon 
     
     
         22 . A substrate onto which one or more of the nucleotide sequences as set froth in SEQ ID NO:1 through SEQ ID NO:9112 is deposited in an ordered array comprising a high density matrix pattern for use in detecting the binding of one or more probe nucleic acid sequences. 
     
     
         23 . A substrate onto which one or more of the proteins encoded by the nucleotide sequences as set forth in SEQ ID NO:1 through SEQ ID NO:9112 is deposited in an ordered array comprising a high density matrix pattern for use in detecting either (a) the binding of an antibody specific for one or more of said proteins, or (b) the binding of one or more peptides that bind specifically to one or more of said proteins. 
     
     
         24 . A nucleic acid array comprising at least one nucleic acid sequence as set forth in SEQ ID NO:1 through SEQ ID NO:9112 stably bound to a solid support. 
     
     
         25 . An array comprising at least one polypeptide encoded by a nucleic acid sequence as set forth in SEQ ID NO:1 through SEQ ID NO:9112 stably bound to a solid support.