Cloning And Sequence Analysis Of NAChR Subunit Gene α6 And Potassium Ion Channel Subunit Gene Shaker From Honeybee, Apis Mellifera And Silkworm, Bombyx Mori

The maturity of eukaryote mRNA needs complicated posttranscriptional processing, such as modification, isoforming, editing, splicing and so on. A-to-I RNA editing is one type of RNA editing which is mediated by Adenosine deaminases that act on RNA. Adenosine deaminases that act on RNA (ADARs) are RNA editing enzymes that change adenosines to inosines via a hydrolytic deamination reaction on double-stranded RNA. Inosine (I) is translated as a guanosine (G), and most enzymes recognize inosine as guanosine. Thus, ADARs change the primary sequence information in a RNA molecule.In this study, the cloning and characterization of nAChR subunitα6 from honeybee(Apis mellifera) and silkworm(Bombyx mori) were reported, which are based on the former research in fly(Drosophila melanogaster) and mosquito(Anopheles gambiae). Using the BLAST algorithm with full coding region and the exons which editing sites located , their orthologues in the honeybee and silkworm genome were identified, and specific primers were designed followed the sequences. Whole RNA was isolated from embryo, larvae, pupae, and adult respectively. Genomic DNA was extracted from adult. Reverse transcription-polymerase chain reaction (RT-PCR) was performed and the PCR products were subjected to direct sequence analysis without coloning. Compared to the sequences of cDNA and genomic DNA, A to G discrepancies between cDNAs and the corresponding genomic sequence were found. Further analysis indicated that some of these sites were A-to-I editing sites. Ultimately, 8 A-to-I editing sites from honeybee and 5 sites from silkworm were found. 3 alternative exon versions of exon8 from honeybee were also found. Simultaneously, further analyzing of the sequences showed some interesting relationship between editing and alternative splicing. Formation of phylogenetic tree through protein sequences alignment of nine insects showed that the evolution ofα6 was conservative.Ion channels are crucial in exitation of nerves, muscles and other tissue membranes. AChRs are classified to ligand gating ion channel. The current research in this paper helps to develop new model of gene expression regulation.