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.
Highlights
Ryanodine receptors (RyRs) are members of a superfamily of intracellular Ca2+ channels
The amino acid sequences alignment showed that the identities of OfRyR with Cnaphalocrocis medinalis RyR (CmRyR), Chilo suppressalis RyR (CsRyR), Spodoptera exigua RyR (SeRyR), Plutella xylostella RyR (PxRyR), Bombyx mori RyR (BmRyR) and Drosophila mojavensis RyR (DmRyR) were 97, 96, 95, 92, 95 and 79%, respectively
L Guo et al cloned two PxRyR cDNA, PxRyR1 and PxRyR2 [31], they are respectively 47 and 91 amino acids shorter than the PxRyR cloned by Wang et al And LN Sun et al reported a PxRyR showing 99% amino acids identity with Wang’s RyR [23]
Summary
Ryanodine receptors (RyRs) are members of a superfamily of intracellular Ca2+ channels. These channels regulate the release of calcium from the lumen of sarcoplasmic/endoplasmic reticulum to the cytosol of muscle and non-muscle cells [1,2]. There is approximately 66% amino acid sequence homology among the three mammalian RyR isoforms, except for three regions with high degrees of variability [9]. Two isoforms (RyRA and RyRB) were found in fish, amphibians and birds [10,11] These two isoforms showed homology with mammals RyR1 and RyR3. The regions with a high degree of structural divergence between the mammalian and insect isoforms of the RyR may serve as potential targets for insecticides that interact with the insect but not mammalian isoforms [9]
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