Molecular Cloning, Characterization and Expression Profiling of a Ryanodine Receptor Gene in Asian Corn Borer, Ostrinia furnacalis (Guenée)

Abstract

Ryanodine receptor (RyR) Ca2+ release channel is the target of diamide insecticides, which show selective insecticidal activity against lepidopterous insects. To study the molecular mechanisms underlying the species-specific action of diamide insecticides, we have cloned and characterized the entire cDNA sequence of RyR from Ostrinia furnacalis (named as OfRyR). The OfRyR mRNA has an Open Reading Frame of 15324 bp nucleotides and encodes a 5108 amino acid polypeptide that displays 79-97% identity with other insects RyR proteins and shows the greatest identity with Cnaphalocrocis medinalis RyR (97%). Quantitative real-time PCR showed that the OfRyR was expressed at the lowest level in egg and the highest level in adult. The relative expression level of OfRyR in first, third and fifth-instar larva were 1.28, 1.19 and 1.99 times of that in egg. Moreover, two alternative splicing sites were identified in the OfRyR gene. One pair of mutually exclusive exons (a/b) were present in the central part of the predicted SPRY domain, and an optional exon (c) was located between the third and fourth RyR domains. Diagnostic PCR demonstrated that exons a and b existed in all developmental stages of OfRyR cDNA, but exon c was not detected in the egg cDNA. And the usage frequencies of these exons showed a significant difference between different developmental stages. These results provided the crucial basis for the functional expression of OfRyR and for the discovery of compound with potentially selective insect activtity. © 2013 Cui et al.