A SYNTHETIC CRYIC GENE, ENCODING A BACILLUS-THURINGIENSIS DELTA-ENDOTOXIN, CONFERS SPODOPTERA RESISTANCE IN ALFALFA AND TOBACCO

Spodoptera species, representing widespread polyphagous insect pests, are resistant to Bacillus thuringiensis delta-endotoxins used thus far as insecticides in transgenic plants, Here we describe the chemical s ynthesis of a cryIC gene by novel template directed ligation-PCR metho d, This simple and economical method to construct large synthetic gene s can be used when routine resynthesis of gents Is required, Chemicall y phosphorylated adjacent oligonucleotides of the gene to be synthesiz ed are assembled and ligated on a single-stranded, partially homologou s template derived from a wild-type gene (cryIC in our case) bg a ther mostable Pfu DNA ligase using repeated cycles of melting, annealing, a nd ligation. The resulting synthetic DN4 strands,Ire selectively ampli fied bg PCR with short specific flanking primers that are complementar y only to the new synthetic DNA. Optimized expression of the synthetic cryIC gene in alfalfa and tobacco results in the production of 0.01-0 .2% of total soluble proteins as CryIC toxin and provides protection a gainst the Egyptian cotton leafworm (Spodoptera littoralis) and the be et armyworm (Spodoptera exigua). To facilitate selection and breeding of Spodoptera-resistant plants, the cryIC gene was linked to a prtf ge ne, conferring resistance to the herbicide BASTA.