A Synthetic cryIC Gene, Encoding a Bacillus thuringiensis δ -endotoxin, Confers Spodoptera resistance in Alfalfa and Tobacco

• Published in: Proceedings of the National Academy of Sciences - PNAS 1996-12, Vol.93 (26), p.15012-15017
• Main Authors: Strizhov, Nicolai, Keller, Menachem, Mathur, Jaideep, Koncz-Kalman, Zsuzsanna, Bosch, Dirk, Prudovsky, Evgenia, Schell, Jeff, Sneh, Baruch, Koncz, Csaba, Zilberstein, Aviah
• Format: Article
• Language: English
• Subjects: Alfalfa; Amino Acid Sequence; Animals; Arabidopsis - physiology; Bacillus thuringiensis Toxins; Bacterial Proteins - biosynthesis; Bacterial Proteins - genetics; Bacterial Toxins; Base Sequence; Biological Sciences; Centrum voor Plantenveredelings- en Reproduktieonderzoek; DNA; DNA Primers; Endotoxins - biosynthesis; Endotoxins - genetics; Genes, Bacterial; Genes, Synthetic; Genetic vectors; Hemolysin Proteins; Insect larvae; Leaves; Medicago sativa - microbiology; Medicago sativa - physiology; Molecular Sequence Data; Moths; Nicotiana - microbiology; Nicotiana - physiology; Oligonucleotides; Pest Control, Biological; Plants; Plants, Genetically Modified; Plants, Toxic; Polymerase Chain Reaction; Recombinant Proteins - biosynthesis; Spodoptera; Toxins; Transgenic plants
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.93.26.15012

Spodoptera species, representing widespread polyphagous insect pests, are resistant to Bacillus thuringiensis δ -endotoxins used thus far as insecticides in transgenic plants. Here we describe the chemical synthesis of a cryIC gene by a novel template directed ligation-PCR method. This simple and economical method to construct large synthetic genes can be used when routine resynthesis of genes is required. Chemically phosphorylated adjacent oligonucleotides of the gene to be synthesized are assembled and ligated on a single-stranded, partially homologous template derived from a wild-type gene (cryIC in our case) by a thermostable Pfu DNA ligase using repeated cycles of melting, annealing, and ligation. The resulting synthetic DNA strands are selectively amplified by PCR with short specific flanking primers that are complementary 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 against the Egyptian cotton leafworm (Spodoptera littoralis) and the beet armyworm (Spodoptera exigua). To facilitate selection and breeding of Spodoptera-resistant plants, the cryIC gene was linked to a pat gene, conferring resistance to the herbicide BASTA.