Chimeric Investigations into the Diamide Binding Site on the Lepidopteran Ryanodine Receptor.

Alterations to amino acid residues G4946 and I4790, associated with resistance to diamide insecticides, suggests a location of diamide interaction within the pVSD voltage sensor-like domain of the insect ryanodine receptor (RyR). To further delineate the interaction site(s), targeted alterations were made within the same pVSD region on the diamondback moth (Plutella xylostella) RyR channel. The editing of five amino acid positions to match those found in the diamide insensitive skeletal RyR1 of humans (hRyR1) in order to generate a human-Plutella chimeric construct showed that these alterations strongly reduce diamide efficacy when introduced in combination but cause only minor reductions when introduced individually. It is concluded that the sites of diamide interaction on insect RyRs lie proximal to the voltage sensor-like domain of the RyR and that the main site of interaction is at residues K4700, Y4701, I4790 and S4919 in the S1 to S4 transmembrane domains.

Extension of Partial Gene Transcripts by Iterative Mapping of RNA-Seq Raw Reads.

Many non-model organisms lack reference genomes and the sequencing and de novo assembly of an organism's transcriptome is an affordable means by which to characterize the coding component of its genome. Despite the advances that have made this possible, assembling a transcriptome without a known reference usually results in a collection of full-length and partial gene transcripts. The downstream analysis of genes represented as partial transcripts then often requires further experimental work in the laboratory in order to obtain full- length sequences. We have explored whether partial transcripts, encoding genes of interest present in de novo assembled transcriptomes of a model and non-model insect species, could be further extended by iterative mapping against the raw transcriptome sequencing reads. Partial sequences encoding cytochrome P450s and carboxyl/cholinesterase were used in this analysis because they are large multigene families and exhibit significant variation in expression. We present an effective method to improve the continuity of partial transcripts in silico that, in the absence of a reference genome, maybe a quick and cost-effective alternative to their extension by laboratory experimentation. Our approach resulted in the successful extension of incompletely assembled transcripts, often to full length. We experimentally validated these results \textit{in silico} and using real-time PCR and sequencing.