Spodoptera Litura Tolerant Transgenic Tobacco Plants Expressing Genetically Modified CRY 1AB Genes from Bacillus Thuringiensis

ABSTRACT

The 130 kD crystal protein coded on Cry 1Ab from Bacillus thuringiensis ssp.aizawai is highly insecticidal against Plutella xylostella larvae and less toxic to Spodoptera litura larvae. The mutant with triple amino acid replacements at Arg₆₁₉, Lys₆₂₂ and Lys₆₃₇ with Gin was 2.5 times more active against S. litura than the wild type protein. The truncated mutant and wild type cry 1Ab genes coding for the first 647 amino acids were each placed under the control of CaMV 35S promoter and Nos terminator in a binary vector, and were transformed into a haploid clone of N. tabacum cv. Samson nn via Agrobacterium. Presence of the transgene in the genomic DNA of tobacco plants was confirmed by PCR and Southern blot. Insecticidal activity of the transgenic plants was examined by infesting freshly hatched S. litura larvae on leaf disks from in vitro grown plants. The biotests showed enhanced protection of the transgenic tobacco plants expressing each of the mutant and wild type Cry 1Ab protein compared to the untransformed control. However, the expression level of the Cry 1Ab active fragment in the leaf tissue was below the detection limit by Western blot even in the plants with the highest insecticidal activity. RT-PCR experiments revealed splicing of cry 1Ab genes at cryptic splicing sites which could not be predicted by sequence analyses.