Identification of functional enolase genes of the silkworm Bombyx mori from public databases with a combination of dry and wet bench processes

Abstract

BackgroundVarious insect species have been added to genomic databases over the years. Thus, researchers can easily obtain online genomic information on invertebrates and insects. However, many incorrectly annotated genes are included in these databases, which can prevent the correct interpretation of subsequent functional analyses. To address this problem, we used a combination of dry and wet bench processes to select functional genes from public databases.ResultsEnolase is an important glycolytic enzyme in all organisms. We used a combination of dry and wet bench processes to identify functional enolases in the silkworm Bombyx mori (BmEno). First, we detected five annotated enolases from public databases using a Hidden Markov Model (HMM) search, and then through cDNA cloning, Northern blotting, and RNA-seq analysis, we revealed three functional enolases in B. mori: BmEno1, BmEno2, and BmEnoC. BmEno1 contained a conserved key amino acid residue for metal binding and substrate binding in other species. However, BmEno2 and BmEnoC showed a change in this key amino acid. Phylogenetic analysis showed that BmEno2 and BmEnoC were distinct from BmEno1 and other enolases, and were distributed only in lepidopteran clusters. BmEno1 was expressed in all of the tissues used in our study. In contrast, BmEno2 was mainly expressed in the testis with some expression in the ovary and suboesophageal ganglion. BmEnoC was weakly expressed in the testis. Quantitative RT-PCR showed that the mRNA expression of BmEno2 and BmEnoC correlated with testis development; thus, BmEno2 and BmEnoC may be related to lepidopteran-specific spermiogenesis.ConclusionsWe identified and characterized three functional enolases from public databases with a combination of dry and wet bench processes in the silkworm B. mori. In addition, we determined that BmEno2 and BmEnoC had species-specific functions. Our strategy could be helpful for the detection of minor genes and functional genes in non-model organisms from public databases.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3455-y) contains supplementary material, which is available to authorized users.

Reference transcriptome data in silkwormBombyx mori

“…We calculated the abundance of each transcript in ten tissues: FB, MG, MT, TT, OV, ASG, MSG-A, MSG-M, MSG-P, PSG, TT, and OV. In comparisons of our calculated results and evaluations of the reliability of our expression analysis, we additionally quantified transcript expression by using public RNA-seq data that were sequenced from the FB, MG, MT, TT, and SG of the B. mori o751 strain (Kikuchi et al, 2017; Ichino et al, 2018; Kobayashi et al, 2019). To distinguish between our RNA-seq data and public data, we referred to the FB, MG, MT, TT, and SG of the o751 strain as BN_FB, BM_MG, BN_ MT, BM_TT, and BM_SG, respectively.…”

“…To estimate the expression of the reference transcriptome in 30 samples, the raw fastq data of each sample and reference transcript data were used with Kallisto ver0.44.0 (Bray et al, 2016). In comparisons of transcriptome data, the raw RNA-seq data of multiple tissues in B. mori strain o751 from the Sequence Read Archive (SRA) and reference transcript data were used: the accession numbers of raw RNA-seq data are DRA005094, DRA005878 and DRA005094 (Kikuchi et al, 2017; Ichino et al, 2018; Kobayashi et al, 2019).…”

Evaluating the potential of direct RNA nanopore sequencing: Metatranscriptomics highlights possible seasonal differences in a marine pelagic crustacean zooplankton community

“…Enolase is a highly conserved metalloenzyme, in both pro-and eukaryotes, with an absolute requirement for magnesium as the natural cofactor (cf. GO term magnesium ion binding, GO:0000287) (Kikuchi et al, 2017). Enolase has at least two important functions in arthropods: (1) gluconeogenesis, the generation of glucose molecules and (2) glycolysis, the first fundamental metabolic process of aerobic respiration, which is responsible for the majority of energy (ATP) production in all organisms (Kikuchi et al, 2017).…”

“…GO term magnesium ion binding, GO:0000287) (Kikuchi et al, 2017). Enolase has at least two important functions in arthropods: (1) gluconeogenesis, the generation of glucose molecules and (2) glycolysis, the first fundamental metabolic process of aerobic respiration, which is responsible for the majority of energy (ATP) production in all organisms (Kikuchi et al, 2017). In addition, arthropod (especially insect) enolases can have species-specific roles as well (Kikuchi et al, 2017).…”

“…Enolase has at least two important functions in arthropods: (1) gluconeogenesis, the generation of glucose molecules and (2) glycolysis, the first fundamental metabolic process of aerobic respiration, which is responsible for the majority of energy (ATP) production in all organisms (Kikuchi et al, 2017). In addition, arthropod (especially insect) enolases can have species-specific roles as well (Kikuchi et al, 2017). Fructose-bisphosphate aldolase is an enzyme encoded by just one gene and is involved in functional gluconeogenic and glycolytic pathways as well (Guo et al, 2012).…”