Characterization of an elm chitinase gene as a possible resistance agent of grasses against insects

The purpose of this research project was to investigate the possible role of plant chitinase in resistance against phytophagous insects. A total of 17 cDNA clones that hybridized with a WIN6 poplar chitinase gene probe were obtained from an elm cDNA library. The clone designated pHS2 was most complete; so it was chosen for more detailed study. The gene was characterized and found to be highly homologous with other plant chitinases. This elm chitinase contains a chitinase catalytic region and a chitin binding domain, and is a typical class I chitinase.; The hs2 elm chitinase gene contains 1225 nucleotides, with a 951 nucleotide open wading frame (ORF). Using the deduced 317-amino acid sequence from this ORF, a homology search using TFastA showed 56 of the 57 best matches to be with chitinases. It has strong homology to broad bean, poplar, and tobacco class I chitinases. The predicted protein product was named ECH2 (elm chitinase 2).; The hs2 deduced amino acid sequence contains two chitinase class I signatures and a chitin binding domain signature. The first signature contains one of the six cysteines conserved in most chitinases. The second chitinase signature is at position 215 in ECH2. A chitin recognition or binding domain signature is found in ECH2 at position 33. The first 21 residues of ECH2 comprise a hydrophobic signal peptide. Residues 312–317 may be a C-terminal extension routing peptide to direct the protein into vacuoles. Residues 22–311 compose the main, catalytic domain.; The hs2 gene was expressed in Escherichia coli . ECH2 from E. coli was injected into a rabbit to produce an antiserum.; A plasmid, designated pKYLX71-hs2, was constructed for plant transformations with hs2. It contained hs2 controlled by the CaMV 35S promoter, a kanamycin resistance gene for selection of putatively transformed plants, a tetracycline resistance gene for selection in bacteria, and T-DNA borders for integration into plant chromosomes via Agrobacterium tumefaciens mediated plant transformation. Tobacco transformed by pKYLX71-hs2 expressed the hs2 gene. Creeping bentgrass transformed by pKYLX71-hs2 via a biolistic method also expressed the hs2 gene.; Transgenic turfgrass plants expressing the hs2 gene were used in bioassays to study the potential of chitinase for plant resistance to insects. Feeding bioassays were done with Japanese beetle larvae. There were very few statistically significant differences (P = 0.05) between the larval growth rates, pupal weights, survival, or pupation of insects that fed on Penncross nontransgenic turfgrass, a transgenic turfgrass line expressing the bar herbicide resistance gene but not the hs2 gene, or four different transgenic turfgrass lines expressing both bar and hs2.