A gut-specific chitinase gene was cloned from the mulberry longicorn beetle, Apriona germari. The A. germari chitinase (AgChi) gene spans 2894 bp and consists of five introns and six exons coding for 390 amino acid residues. AgChi possesses the chitinase family 18 active site signature and three N-glycosylation sites. Southern blot analysis of genomic DNA suggests that AgChi is a single copy gene. The AgChi cDNA was expressed as a 46-kDa polypeptide in baculovirus-infected insect Sf9 cells and the recombinant AgChi showed activity in a chitinase enzyme assay. Treatment of recombinant virus-infected Sf9 cells with tunicamycin, a specific inhibitor of N-linked glycosylation, revealed that AgChi is N-glycosylated, but the carbohydrate moieties are not essential for chitinolytic activity. Northern and Western blot analyses showed that AgChi was specifically expressed in the gut; AgChi was expressed in three gut regions, indicating that the gut is the prime site for AgChi synthesis in A. germari larvae.
Transgenic lines of silver birch (Betula pendula) carrying the sugar beet chitinase IV gene were used to study the effects of the heterologous expression of a transgenic chitinase on the performance of lepidopteran herbivores. The effect of wounding the leaves of birch on the performance of lepidopteran larvae and the growth of trees was also studied. Larvae of Orgyia antiqua L., Lymantriidae, and Phalera bucephala L., Notodontidae, were separately fed on the leaves of transgenic and wild-type birch, and their performance measured using nutritional indices. The relative growth rate (RGR) of O. antiqua larvae fed transgenic leaves was significantly lower than that of larvae fed wild-type leaves. Furthermore, there is little evidence that transgenic chitinase affects survival but it was lowest for the group of larvae fed leaves with the highest expression of chitinase IV. Wounding did not have a significant effect on the performance of the larvae or on the growth of the branches of the trees. The growth of branches of particular transgenic lines, however, was significantly associated with tree line. The performance of P. bucephala larvae fed leaves of transgenic and wild-type birches did not differ. The leaves used in both experiments from transgenic trees were shorter than those from wild-type trees. Using transgenic birch expressing sugar beet chitinase IV to improve the resistance of birch to fungal diseases can have negative effects on O. antiqua larvae feeding on the leaves of these birches. P. bucephala, however, was not similarly affected, which indicates that these two ecologically similar lepidopteran species may differ in their response to transgenic chitinase., Liisa Vihervuori ... [et al.]., and Obsahuje seznam literatury