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The insecticidal crystal protein gene of the coleopteran-toxic Bacillus thuringiensis var. tenebrionis has been isolated, and the nucleotide sequence has been determined. A total DNA library from var. tenebrionis was made in the plasmid vector pUC12. By using a synthetic 27-base oligonucleotide corresponding to a stretch of nine N-terminal amino acids of a tryptic fragment of purified crystal protein of var. tenebrionis as a probe, recombinant colonies were screened by in situ hybridization for the presence of the crystal protein gene. Positive clones obtained from this screening were further tested for toxicity. One recombinant, NSBP544 (which contained a 5.9-kilobase BamHI insert), was toxic to larvae of Colorado potato beetle. Immunoblot analysis revealed that this clone produces two crystal-specific antigens of 65 and 73 kDa as do sporulating var. tenebrionis cells. However, purified crystal inclusions from var. tenebrionis contain a primary peptide component of 65 kDa. A 1932-base-pair open reading frame with a coding capacity of 73,119 Da has been identified by nucleotide sequencing analysis of the cloned crystal protein.In addition, mung bean nuclease mapping indicates that transcription of the crystal protein of var. tenebrionis initiates 130 base pairs upstream from the translational start site. Southern blot analysis using an internal 0.7-kilobase EcoRI fragment of pNSBP544 as a probe revealed that the crystal protein gene is located on a 90-MDa plasmid.Bacillus thuringiensis is unique in its ability to produce proteinaceous, crystalline inclusions (8 endotoxins) during the process of sporulation, which are found to be highly toxic to larvae of several lepidopteran insect pests of agricultural importance (1). Numerous isolates of B. thuringiensis strains belonging to over two dozen distinct flagellar serotypes have been isolated and classified to date (2). The crystal proteins of several of these varieties have a rather narrow host range and hence are used commercially as very selective biological insecticides. Strains of B. thuringiensis that are toxic to larvae of dipteran insects have also been isolated recently (3,4).The 3-endotoxin genes of various B. thuringiensis varieties have been cloned by several research groups (5-10). Kronstad et al. (11) have shown that these genes are generally located on one or more large plasmids. These authors have also demonstrated that while several lepidopteran-active genes share extensive nucleotide sequence similarity, no homology exists between the lepidopteran and dipteran toxin genes.Since several agronomically important insect pests belong to the order Coleoptera (e.g., Colorado potato beetle, boll weevil, and corn root worm), an extensive search for coleopteran-toxic B. thuringiensis strains has been undertaken by several laboratories. Such efforts have resulted in the recent isolation oftwo strains ofB. thuringiensis, namely var. tenebrionis (12) Peptide Analysis and Oligonucleotide Synthesis. Crystal inclusions were purified from a crude spore/cr...

We have examined expression of several insecticidal crystal protein (ICP) genes of Bacillus thuringiensis in transgenic tobacco plants and electroporated carrot protoplasts. We determined that low levels of lepidopteran toxin cryIA(b) ICP gene expression in plants and electroporated carrot cells is due to RNA instability. We used a series of 3' deleted by cryIA(b) constructs directed by the cauliflower mosaic virus 35S promoter to demonstrate that this instability is minimally contained in the first 579 bases of the gene in both systems. This instability may result from 5'----3' as well as 3'----5' RNA metabolism. The coleopteran toxic cryIIIA gene was also examined in electroporated carrot cells, and found to be poorly expressed. A model for improvement of ICP RNA stability in plants is presented.

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