Genome-Wide Identification, Characterization and Expression Analysis of the Solute Carrier 6 Gene Family in Silkworm (Bombyx mori)

Abstract

The solute carrier 6 (SLC6) gene family, initially known as the neurotransmitter transporters, plays vital roles in the regulation of neurotransmitter signaling, nutrient absorption and motor behavior. In this study, a total of 16 candidate genes were identified as SLC6 family gene homologs in the silkworm (Bombyx mori) genome. Spatio-temporal expression patterns of silkworm SLC6 gene transcripts indicated that these genes were highly and specifically expressed in midgut, brain and gonads; moreover, these genes were expressed primarily at the feeding stage or adult stage. Levels of expression for most midgut-specific and midgut-enriched gene transcripts were down-regulated after starvation but up-regulated after re-feeding. In addition, we observed that expression levels of these genes except for BmSLC6-15 and BmGT1 were markedly up-regulated by a juvenile hormone analog. Moreover, brain-enriched genes showed differential expression patterns during wandering and mating processes, suggesting that these genes may be involved in modulating wandering and mating behaviors. Our results improve our understanding of the expression patterns and potential physiological functions of the SLC6 gene family, and provide valuable information for the comprehensive functional analysis of the SLC6 gene family.

Keywords: Bombyx mori; expression pattern; motor behavior; nutrient absorption; the solute carrier 6 (SLC6) gene.

Figure 1

A multiple sequence alignment (MSA) of Bombyx mori the Solute Carrier 6 (SLC6) proteins with A. aeolicus LeuT_Aa. The protein domains, including transmembrane (TM) regions, extracellular linker (EL) regions (blue lines), intracellular linker (IL) regions (pink lines), alpha helical structure (thick black line), and beta sheets (tiny black lines), are annotated at the top of each sequence block. The annotation of protein domains is based on published studies [7,23]. The consensus is absolutely conserved for amino acid residues in the sequence alignment of Yamashita [7]. The background of amino acid residues is based on the degree of conservation (black = 100%, dark gray = 80%, light gray = 60%).

Figure 1

A multiple sequence alignment (MSA) of Bombyx mori the Solute Carrier 6 (SLC6) proteins with A. aeolicus LeuT_Aa. The protein domains, including transmembrane (TM) regions, extracellular linker (EL) regions (blue lines), intracellular linker (IL) regions (pink lines), alpha helical structure (thick black line), and beta sheets (tiny black lines), are annotated at the top of each sequence block. The annotation of protein domains is based on published studies [7,23]. The consensus is absolutely conserved for amino acid residues in the sequence alignment of Yamashita [7]. The background of amino acid residues is based on the degree of conservation (black = 100%, dark gray = 80%, light gray = 60%).

Figure 2

Phylogenetic tree of SLC6 transporters from B. mori and other insect species. Colors represent different subfamilies or branches. NTT: neurotransmitter transporter, AAT: amino acid transporter, INE: inebriated gene, NAT: nutrient amino acid transporter, Orphan: orphan transporter. SLC6 transporters from D. melanogaster (Dm), A. gambiae (Ag), and A. aegypti (Ae), whose genome sequences have been reported [29,30,31], were selected to characterize the evolutionary relationships of insect SLC6 transporters. B. mori SLC6 transporters are highlighted in boldface.

Figure 3

The tissue expression profiles of silkworm SLC6 family genes on third day of the fifth instar. The cDNA templates were derived from ovaries (Ov), testis (Te), head (He), cuticles (Cu), fat body (Fb), midgut (Mg), hemocytes (Hc), Malpighian tubules (Mt), and silk gland (Sg).

Figure 4

Analysis of temporal expression patterns of midgut-specific and midgut-enriched SLC6 members. M: molting larvae; N: newly molted larvae; 2d5I, 4d5I, and 6d5I: Days 2, 4, and 6 of the fifth instar, respectively; WS: wandering stage. * p < 0.05, ** p < 0.01 and *** p < 0.001. ns: no significant differences (p > 0.05).

Figure 5

Expression levels of brain-enriched genes in mating and wandering behavior. Error bars indicate the standard error of the mean (n = 3). 3d5I: feeding stage; PP: before pupation; P-1d: the first day after pupation; P-5d: the fifth day after pupation; Moth: newly molted moth. * p < 0.05, ** p < 0.01 and *** p < 0.001.

Figure 6

Influences of starvation on the expression of midgut-specific and midgut-enriched SLC6 genes. F: feeding; S: starvation; SR: starvation and re-feeding. Significant differences were assessed by a student’s t-test (* p < 0.05, ** p < 0.01 and *** p < 0.001). Error bars indicate the standard error of the mean (n = 3).

Figure 7

Induced expression of midgut-specific and midgut-enriched SLC6 genes by juvenile hormone analog (JHA) in ex-vivo midgut. Significant differences were assessed by a student’s t-test (* p < 0.05, ** p < 0.01 and *** p < 0.001). Error bars indicate the standard error of the mean (n = 3). CK: control group; JHA: juvenile hormone analog.