Patent
Disclosed is a novel δ-endotoxin, designated CryET29, that exhibits insecticidal activity against siphonapteran insects, including larvae of the cat flea ( Ctenocephalides felis ), as well as against coleopteran insects, including the southern corn rootworm ( Diabrotica undecimpunctata ), western corn rootworm ( D. virgifera ), Colorado potato beetle ( Leptinotarsa decemlineata ), Japanese beetle ( Popillia japonica ), and red flour beetle ( Tribolium castaneum ). Also disclosed are nucleic acid segments encoding CryET29, recombinant vectors, host cells, and transgenic plants comprising a cryET29 DNA segment. Methods for making and using the disclosed protein and nucleic acid segments are disclosed as well as assays and diagnostic kits for detecting cryET29 and CryET29 sequences in vivo and in vitro.
1. An isolated nucleic acid segment characterized as:
(a) a nucleic acid segment consisting of at least 30 nucleotides that has the same sequence as, or is fully complementary to, 30 contiguous nucleotides of SEQ ID NO:1; or
(b) a nucleic acid segment consisting of at least 30 nucleotides that specifically hybridizes to SEQ ID NO:1 or the full complement thereof under stringent conditions for high selectivity comprising 0.02 M to 0.15 M NaCl at temperatures of about 50° C. to about 70° C.
2. The isolated nucleic acid segment of claim 1 consisting of the sequence as set forth in SEQ ID NO:4.
3. The isolated nucleic acid segment of claim 1 comprising the sequence as set forth in SEQ ID NO:4.
4. A method for detecting a nucleic acid sequence encoding a Coleopteran toxic crystal protein, comprising the steps of:
(a) obtaining sample nucleic acids suspected of encoding a Coleopteran toxic crystal protein;
(b) contacting said sample nucleic acids with the isolated nucleic acid segment of claim 1 , 2 or 3 under conditions effective to allow hybridization of complementary nucleic acids; and
(c) detecting the hybridized complementary nucleic acids thus formed.
5. The method of claim 4 , wherein said nucleic acid segment comprises a detectable label and said hybridized complementary nucleic acids are detected by detecting said label.
6. A nucleic acid detection kit comprising, in suitable container means, the nucleic acid segment of claim 1 , 2 or 3 and a detection reagent.