title: Identification of functional enolase genes of the silkworm Bombyx mori from public databases with a combination of dry and wet bench processes.
abstract: Various 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.
  
  
  
  Enolase 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.
  
  
  
  We 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.
authors.affiliation: Department of Science of Biological Production, Graduate School of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo, 183-8509, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Kikuchi
authors.affiliation: Database Center for Life Science (DBCLS), Joint Support-Center for Data Science Research, Research Organization of Information and Systems (ROIS), Yata 1111, Mishima, Shizuoka, 411-8540, Japan.
authors.forename: Takeru
authors.initials: T
authors.lastname: Nakazato
authors.affiliation: Department of Science of Biological Production, Graduate School of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo, 183-8509, Japan.
authors.forename: Katsuhiko
authors.initials: K
authors.lastname: Ito
authors.affiliation: Department of Science of Biological Production, Graduate School of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo, 183-8509, Japan.
authors.forename: Yosui
authors.initials: Y
authors.lastname: Nojima
authors.affiliation: Department of Science of Biological Production, Graduate School of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo, 183-8509, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Yokoyama
authors.affiliation: Department of Bioregulation and Biointeraction, Graduate School of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo, 183-8509, Japan.
authors.forename: Kikuo
authors.initials: K
authors.lastname: Iwabuchi
authors.affiliation: Database Center for Life Science (DBCLS), Joint Support-Center for Data Science Research, Research Organization of Information and Systems (ROIS), Yata 1111, Mishima, Shizuoka, 411-8540, Japan.
authors.forename: Hidemasa
authors.initials: H
authors.lastname: Bono
authors.affiliation: Center for Information Biology, National Institute of Genetics, Yata 1111, Mishima, Shizuoka, 411-8540, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Toyoda
authors.affiliation: Center for Information Biology, National Institute of Genetics, Yata 1111, Mishima, Shizuoka, 411-8540, Japan.
authors.forename: Asao
authors.initials: A
authors.lastname: Fujiyama
authors.affiliation: Graduate School of Bio-Applications and Systems Engineering (BASE), 2-24-16, Naka-cho, Koganei, Tokyo, 184-8588, Japan.
authors.forename: Ryoichi
authors.initials: R
authors.lastname: Sato
authors.affiliation: Department of Science of Biological Production, Graduate School of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo, 183-8509, Japan. h_tabuno@cc.tuat.ac.jp.
authors.forename: Hiroko
authors.identifier: 0000-0003-1823-9952
authors.initials: H
authors.lastname: Tabunoki
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D012831:Bombyx / Q000235:genetics*; D030561:Databases, Nucleic Acid; D004777:Environment; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D017344:Genes, Insect; D059014:High-Throughput Nucleotide Sequencing; D009928:Organ Specificity / Q000235:genetics; D010751:Phosphopyruvate Hydratase / Q000737:chemistry / Q000235:genetics*; D059467:Transcriptome
chemical_list: D010751:Phosphopyruvate Hydratase
keywords: Analysis pipeline; Bombyx mori; Enolase; Growth regulator; Reproduction