Molecular characterization and alternative splicing of a sodium channel and DSC1 ortholog genes in Liposcelis bostrychophila Badonnel (Psocoptera: Liposcelididae)

Abstract

Alternative splicing greatly contributes to the structural and functional diversity of voltage-gated sodium channels (VGSCs) by generating various isoforms with unique functional and pharmacological properties. Here, we identified a new optional exon 23 located in the linker between domains II and III, and four mutually exclusive exons (exons 27A, 27B, 27C, and 27D) in domains IIIS3 and IIIS4 of the sodium channel of Liposcelis bostrychophila (termed as LbVGSC). This suggested that more alternative splicing phenomena remained to be discovered in VGSCs. Inclusion of exon 27C might lead to generation of non-functional isoforms. Meanwhile, identification of three alternative exons (exons 11, 13A, and 13B), which were located in the linker between domains II and III, indicated that abundant splicing events occurred in the DSC1 ortholog channel of L. bostrychophila (termed as LbSC1). Exons 13A and 13B were generated by intron retention, and the presence of exon 13B relied on the inclusion of exon 13A. Exon 13B was specifically expressed in the embryonic stage and contained an in-frame stop codon, inclusion of which led to generation of truncated proteins with only the first two domains. Additionally, several co-occurring RNA editing events were identified in LbSC1. Furthermore, remarkable similarity between the structure and expression patterns of LbVGSC and LbSC1 were discovered, and a closer evolutionary relationship between VGSCs and DSC1 orthologs was verified. Taken together, the data provided abundant molecular information on VGSC and DSC1 orthologs in L. bostrychophila, a representative Psocoptera storage pest, and insights into the alternative splicing of these two channels.

Keywords: Alternative splicing; DSC1; Liposcelis bostrychophila; RNA editing.; VGSC; qPCR.

Fig 1

Strategies used in cloning LbVGSC and LbSC1 and schematic representation of topology of VGSCs and DSC1 orthologs. The four homologous domains (I-IV), each having six transmembrane fragments (S1-S6), were indicated. Fragments “m1”, “m2”, “n1”, and “n2” were derived from the transcriptome data of L. bostrychophila (SRS390072). Fragments “a”, “b”, “c”, “d”, and “e” were used to assemble LbVGSC, and “a1”, “b1”, and “c1” were used to assemble LbSC1. Lines indicated the length and relative location of amplicons in relation to the topological structure. The solid boxes indicated the alternatively spliced exons. The solid triangle showed the inactivation gate, labeled with MFM/MFL.

Fig 2

Alignments of protein sequences of LbVGSC, LbSC1, para, DSC1, para^CSMA, BSC1, PhVGSC (para ortholog from Pediculus humanus), and PhSC1 (DSC1 ortholog from P. humanus). The GenBank accession numbers were indicated following the sequence names. The transmembrane segments (S1-S6) were underlined for LbVGSC and highlighted in black for LbSC1. The four-amino-acid DEKA motif in VGSCs and DEEA in DSC1 orthologs, for ion selection, in each linker between S5 and S6, were highlighted in black in each S4 region. The inactivation gates “MFM” and “MFL” between II and III were highlighted and boxed. All alternatively spliced exons included in the sequences were boxed, and thin arrows indicated the positions of those not included. The PKC sites were denoted with bold arrows. PKA sites were denoted with “▼” for LbVGSC and “▲” for LbSC1. The N-glycosylation sites were denoted with “◆”. “*” denoted identical residues; “:” denoted a conservative residue substitution; “.” denoted partial conservation of the residue.

Fig 2

Alignments of protein sequences of LbVGSC, LbSC1, para, DSC1, para^CSMA, BSC1, PhVGSC (para ortholog from Pediculus humanus), and PhSC1 (DSC1 ortholog from P. humanus). The GenBank accession numbers were indicated following the sequence names. The transmembrane segments (S1-S6) were underlined for LbVGSC and highlighted in black for LbSC1. The four-amino-acid DEKA motif in VGSCs and DEEA in DSC1 orthologs, for ion selection, in each linker between S5 and S6, were highlighted in black in each S4 region. The inactivation gates “MFM” and “MFL” between II and III were highlighted and boxed. All alternatively spliced exons included in the sequences were boxed, and thin arrows indicated the positions of those not included. The PKC sites were denoted with bold arrows. PKA sites were denoted with “▼” for LbVGSC and “▲” for LbSC1. The N-glycosylation sites were denoted with “◆”. “*” denoted identical residues; “:” denoted a conservative residue substitution; “.” denoted partial conservation of the residue.

Fig 2

Alignments of protein sequences of LbVGSC, LbSC1, para, DSC1, para^CSMA, BSC1, PhVGSC (para ortholog from Pediculus humanus), and PhSC1 (DSC1 ortholog from P. humanus). The GenBank accession numbers were indicated following the sequence names. The transmembrane segments (S1-S6) were underlined for LbVGSC and highlighted in black for LbSC1. The four-amino-acid DEKA motif in VGSCs and DEEA in DSC1 orthologs, for ion selection, in each linker between S5 and S6, were highlighted in black in each S4 region. The inactivation gates “MFM” and “MFL” between II and III were highlighted and boxed. All alternatively spliced exons included in the sequences were boxed, and thin arrows indicated the positions of those not included. The PKC sites were denoted with bold arrows. PKA sites were denoted with “▼” for LbVGSC and “▲” for LbSC1. The N-glycosylation sites were denoted with “◆”. “*” denoted identical residues; “:” denoted a conservative residue substitution; “.” denoted partial conservation of the residue.

Fig 2

Alignments of protein sequences of LbVGSC, LbSC1, para, DSC1, para^CSMA, BSC1, PhVGSC (para ortholog from Pediculus humanus), and PhSC1 (DSC1 ortholog from P. humanus). The GenBank accession numbers were indicated following the sequence names. The transmembrane segments (S1-S6) were underlined for LbVGSC and highlighted in black for LbSC1. The four-amino-acid DEKA motif in VGSCs and DEEA in DSC1 orthologs, for ion selection, in each linker between S5 and S6, were highlighted in black in each S4 region. The inactivation gates “MFM” and “MFL” between II and III were highlighted and boxed. All alternatively spliced exons included in the sequences were boxed, and thin arrows indicated the positions of those not included. The PKC sites were denoted with bold arrows. PKA sites were denoted with “▼” for LbVGSC and “▲” for LbSC1. The N-glycosylation sites were denoted with “◆”. “*” denoted identical residues; “:” denoted a conservative residue substitution; “.” denoted partial conservation of the residue.

Fig 3

Phylogenetic analysis of 67 insect amino acid sequences, including 29 VGSCs, 22 DSC1 orthologs, and 16 VGCCs. LbVGSC was denoted with a solid circle, and LbSC1was denoted with a solid triangle. Only bootstrap values exceeding 50% were shown at branch points. GenBank accession numbers of all sequences were listed following the species names and in Tables S3-S5. Members of VGSCs, DSC1 orthologs, and VGCCs were highlighted with different colors and denoted with corresponding names.

Fig 4

Developmental and tissue-specific expression of LbVGSC. (A) RT-qPCR analysis of developmental expression patterns from the embryonic to adult stages. Different letters above the bar indicated significant differences in the expression level of LbVGSC (p < 0.05, one-way ANOVA). (B) RT-PCR analysis of LbVGSC in head, thorax, and abdomen of adult insects. Each PCR product (10 μL) was separated on a 2.0% agarose gel.

Fig 5

Developmental and tissue-specific expression of LbSC1. (A) RT-qPCR analysis of developmental expression patterns from embryonic to adult stages. Different letters above the bar indicated significant differences in the expression level of LbSC1 (p < 0.05, one-way ANOVA). (B) RT-PCR analysis of LbSC1 in head, thorax, and abdomen of adult insects. Each PCR product (10 μL) was separated on a 2.0% agarose gel.

Fig 6

Detection of alternatively spliced exons in different developmental stages and tissues by RT-PCR. (A) PCR detection for exons in different developmental stages. Primers were designed to flank the splicing sites. Different bands indicated various products by inclusion or exclusion of specific exons. Each PCR product (10 μL) was separated on a 2.0% agarose gel. (B) PCR detection for exons 13A and 13B. The amplified region included exon 13A and part of exon 13B. The antisense primer (13A/13B-R1) was designed within exon 13B. 20 μL of the product was separated on a 2.0% agarose gel to detect any low-abundance transcripts.

Fig 7

Amino acid sequences, schematic of the genomic structure, and splicing patterns of exon 23 in LbVGSC, and exons 11, 13A and 13B in LbSC1. Amino acid sequences of alternative exons were shown above the nucleotide sequence. The genomic structure of the corresponding exon was indicated below. All exons and introns were indicated with six bases at the junction. The exons were represented by boxed uppercase letters, and retained introns 13A and 13B were indicated by boxed lowercase letters. The adjacent introns were indicated using lowercase letters without boxes. The alternative exons were labeled with corresponding names. Sizes of the bordering introns and alternative exons were indicated. The junctions of exons were indicated with bridge lines. The consensus splice donor and acceptor sequences, gt-ag, were in bold. The arrows showed the relative positions of primers used in PCR. The three nucleotides, TTA, highlighted in gray, were present in the genomic sequence but not in exon 13A. The premature stop codon TAA was denoted with an asterisk in exon 13B.

Fig 8

Genomic sequences, splicing patterns, and amino acid sequence alignments of exons 27A, 27B, 27C, and 27D. (A) The amplified genomic sequences containing exon 27A, 27B, 27C, and 27D. Introns were indicated with lowercase letters. The deduced amino acid sequences of the alternative exons were indicated below the nucleotides. The splicing consensus sequences were highlighted in gray. Exon 27C, located within exon 27B, was underlined. Arrows indicated primers used in PCR. (B) Schematic of splicing patterns. Constitutive exons were indicated by blank boxes, alternative exons were indicated by gray boxes, and introns were indicated by straight lines. The junctions of exons were indicated by bridge lines. The scale was not proportional. (C) Alignments of exons 27A/27B/27C/27D with k/l in para and G1/G2 in para^CSMA. The transmembrane fragment IIIS4 was boxed.