| The insect nicotinic acetylcholine receptor (nAChR) and voltage-gated sodiumchannel (VGSC) are action targets of neonicotinoid and pyrethroid insecticidesrespectively. Lepidoptera is the second largest order of Insecta. Most insects of thisorder are herbivores and eat plants at the larval stage and many are agricultural pests.Studying silkworm nAChR and VGSC will benefit to pest control.The nAChR gene family always has more than ten members and the combinationof the members gives rise to high variability of nAChR subunit composition, whichmakes study of their physiological functions and electrophysiological characteristicsvery difficult. The study of nAChR of lepidopteran insects may provide knowledge tothe design and development of new insecticides. We did following experiments aboutthe nAChR of Lepidoptera model insect, Bombyx mori, silkworm.After BLAST search of the silkworm genome database and by RT-PCR andRACE, 12 full length ORFs coding nAChR subunits were cloned. Silkworm nAChRsubunit genes share high amino acid identities with fruit fly nAChR subunit genes upto 86%. Nine subunits wereαtype and three subunits wereβtype. Only subunitsα6 of silkworm, fruit fly, mosquito and honeybee share identical exon-intronjunction positions.Silkworm nAChR subunitα4 has two exclusively spliced exons 3 and subunitα8 has two exclusively spliced exons 7. The exclusively splicedα8 exons 7encoding TM2 and TM3 were only found in silkworm. Truncated transcriptα4lacks exons 6～8, those exons encode LoopF and TM1-3. Truncated transcriptα5misses exon 4, which gives rise to frameshift and only a short ORF is retained. RNAediting was only detected at subunitα6. Five RNA editing sites (nts #392, 394, 395,447,454) give rise to four amino acid changes (N131S, N132G, I149M, T152A).Expression levels of seven transcripts in four developmental stages wereanalyzed by real-time PCR. All transcripts have highest expression levels at heads andlowest expression levels at abdomens. Compared with larva period, all transcripts have higher expression levels at pupa period and adult period, transcripts ofα2,α8exon7a,α8 exon7b have lower expression levels, transcripts ofα4 exon3,α7,β1 have higher expression levels, andα4 exon3' has equal amount of expression levelat embryonic period.We performed following experiments about silkworm sodium channel:After BLAST search of the silkworm genome database and by RT-PCR, fulllength ORF of silkworm sodium channel gene was cloned and named BmNav. BmNavORF contains 6,258bp, encodes 2,085 amino acid residues and has 34 exons. BmNavshares 74%, 77%, 43% amino acid identities with sodium channel of fruit fly, Germancockroach, and human being. BmNav has 24 transmembrane regions which belong tofour domains, and each domain has six transmembrane regions.BmNav PCR products from four developmental stages including embryo, larva,pupa, and adult, were sequenced by high through-put Solexa technology. None RNAediting was found and a large amount of alternative splicing transcripts were detected.The missed exons not only encode peptides between domains, but also encodepeptides of membrane regions. Optional exons 2, 12, and 22i encode for peptidesbetween the domains, and the conserved regions are not influenced without thoseexons. The expression level of transcripts without exon 2 is high up to 33%～55% infour developmental stages. Exon 12 was only found in silkworm. Two pairs ofexclusively spliced exons, 19a/b and 27a/b, were found in BmNav. Solexa sequencingresult indicated that expression level of transcript without exon 19 was very high inlarva stage. Routine RT-PCR result indicated that the expression level of thistranscript came to climax on 3rd day of 5th instar and went down after that.
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