Cloning And Alternative Splicing Analysis Of Sodium Channel Genes In Helicoverpa Armigera And Tribolium Castaneum

Voltage-gated sodium channels play an important role in the generation and propagation of action potentials in excitable cells, and are primary targets of several insecticides including DDT, pyrethroids and oxadiazines. At present, rsearches on the relationship between insect sodium channels and insecticide resistance mainly focus on the knockdown resistance (kdr) of pyrethroid insecticides. The cotton bollworm, Helicoverpa armigera (Hubner) is an important pest in cotton, and has developed serious resistance to pyrethroids in many countries. The red flour bettele, Tribolium castaneum, is an important pest in stored grain and is the forth pest that is whole-genome sequenced. In this study, the sodium chanmel genes from H. armigera and T. castaneum were cloned, and the genomic structure and alternative splicing of these two genes were also studied. The results of the research might assist in understanding the structure-function relationships of insect sodium channels as well as the development of novel insecticides.The full-length cDNA sequences of voltage-gated sodium channel a-subunits homologous to the para gene from Drosophila melanogaste were cloned from Helicoverpa armigera (named as HaNav) and Tribolium castaneum (TcNav) using RT-PCR. The composite HaNav contains an ORF of6168bp encoding a protein of2055amino acid residues. The ORF of TcNav contains6152bp and encodes2045amino acid residues. An amino acid sequence alignment shows that HaNav and TcNav share76%identity with each other, and are75%identical with D. melanogaster para. Both HaNav and TcNav proteins share homologous overall organization with all known sodium channel subunits, including four large hydrophobic domains, each composed of six membrane-spanning segments. Specifically, the predominantly negatively charged DEKA motif (D, E, K, and A, respectively, in the putative pore positions in the short segments connecting S5and S6of domains I-IV), which forms the ion selectivity filter and is possessed by all functional sodium channel proteins, is completely conserved and positioned in each of the four domains in the HaNav (D385, E997, K1517, A1810) and TcNav (D387, E988, K1492, A1785). The conserved tripeptide MFM motif for fast inactivation in loop3(L3) in both insects and arachnids is also detected in HaNav at positions1584-1586and TcNav1559-1561. Further expression analysis using qRT-PCR showed that the mRNA expression levels of HaNav and TcNav varied among different developmental stages.Genomic structures of HaNav and TcNav were predicted by comparison of the composite cDNA sequences with the genomic sequences. The HaNav was split into33exons including four mutually exclusive exons, which shares nearly identical genomic organization with previously identified NaV from three Lepitopteran species, Heliothis virescens, Plutella xylostella, and Bombyx mori. In comparison to HaNav, the TcNav has26exons, and exons corresponding to8-10,22-25,30-31,32-33in HaNav are combined into single exons. Pairwise comparison showed that the identity between each pair of exons of the HaNav and TcNav genes was43-87%. The length of15exons is strongly conserved in both HaNav and TcNav, while the lengths of introns vary considerably, on average, HaNav introns are4.7times longer than TcNav ones. The5’donor and3’acceptor site sequences in both HaNav and TcNav are in agreement with the GT/AG consensus sequence, except5’donor sequence (GC) for intron12in HaNav.The alignment of multiple cDNA clone sequences verified several alternative splice sites in both HaNav and TcNav. Exon2,3’portion of exon11,5’portion of exon21were found to be optionally included in HaNav (optional exons), which correspond to optional exons j, i and f of para respectively. Two pairs of mutually exclusive exons were found in HaNav including18a/18b and26a/26b, which correspond to mutually exclusive exons c/d and k/1of para, respectively. While optional exons e and h of para were not identified in HaNav, sequences in5’ portion of exon12and16, correspondind to optional exons a and b of para, respectively, were found in all cDNA clones. In TcNav, Exon2,3’portion of exon9were found to be optionally included in transcripts, which correspond to optional exons j and i of para respectively, and sequence in5’portion of exon10, correspondind to optional exon a of para, was found in all cDNA clones. Similar to HaNav, optional exons e and h of para were not identified in TcNav. In contrast to HaNav and para, which have two pairs of mutually exclusive exons, only exons corrending to d and1of para were identified in both cDNA and genomic sequences of TcNav. While the number of alternative splicing sites in TcNav is much lower than that in HaNav, four short introns16,17,18and19were found in several cDNA clones (transcripts), indicating the occurrence of intron retention. Interesting, while the intron16caused the shift of the open reading frame and resulted in premature termination codons (PTC) in the sequence downstream of the alternative exon, the transcripts containing symmetrical introns17,18and19are translated in frame, but retention of intron19leads to in-frame PTC.