International Journal of Industrial Entomology and Biomaterials

Korean Society of Sericultural Science (한국잠사학회)
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The innate immune response transcription factor Bombyx mori Relish1 induces high-level antimicrobial peptides in silkworm

  • Kim, Seong-Wan (Department of Agricultural Biology, National Institute of Agricultural Science, RDA) ;
  • Kim, Seong-Ryul (Department of Agricultural Biology, National Institute of Agricultural Science, RDA) ;
  • Goo, Tae-Won (Department of Biochemistry, School of Medicine, Dongguk University) ;
  • Choi, Kwang-Ho (Department of Agricultural Biology, National Institute of Agricultural Science, RDA)
  • Received : 2018.11.20
  • Accepted : 2018.12.04
  • Published : 2018.12.31
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Abstract

To artificially enhance antimicrobial peptide expression in Bombyx mori, we constructed genetically engineered silkworms overexpressing Rel family transcription factor. The truncated BmRelish1 (BmRelish1t) gene contained a Rel homolog domain (RHD), nuclear localization signal (NLS), acidic and hydrophobic amino acid (AHAA)-rich region, and death domain (DD), but no ankyrin-repeat (ANK) domain. The BmRelish1t gene was controlled by B. mori cytoplasmic actin 3 promoter in the PiggyBac transposon vector. Chromosome analysis of G1 generations of a transgenic silkworm with EGFP expression confirmed stable insertion of BmRelish1t. BmRelish1t gene overexpression in transgenic silkworms resulted in higher mRNA expression levels of B. mori antimicrobial peptides such as lebocin(~20.5-fold), moricin(~8.7-fold), and nuecin(~17.4-fold) than those in normal silkworms.

Keywords

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Fig. 1. Construction of the PG-3xP3EGFP-BmA3BmRelish1t vector and fluorescent image of transgenic silkworms. (A) Schematic of the PG-3xP3EGFP-BmA3BmRelish1t vector. The BmRelish1t gene was inserted into the PiggyBac plasmid between the BmA3 promoter and SV40 poly A. The EGFP gene was used as a transformation marker. (B1) G1 broods with EGFP-positive embryos at the seventh day of embryonic development were viewed under fluorescence. (B2)The G1 third-instar larva was viewed under fluorescence. Arrows in panels 1, 2, point to the ocelli and the abdominal nervous system, respectively. (B3,4) Pupae and adults were also viewed under fluorescence. Arrows in panels 3, 4 point to the compound eyes.

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Fig. 2. Comparison of mRNA levels of the BmRelish1t-inducible antimicrobial peptide genes in transgenic silkworm larvae. (A) Comparison of mRNA levels of the BmRelish1t genes in transgenic silkworm. mRNA quantification was normalized to ribosomal protein RP49 mRNA. Results of triplicate experiments are shown with their standard deviations. (B) mRNA quantification was normalized to ribosomal protein RP49 mRNA. Results of triplicate experiments are shown with their standard deviations. Antimicrobial peptide genes are CecropinD2 (NM_001043459), Enbocin (U30289), Gloverin1 (AB289654), Lebocin (S79612), Moricin (NM_001043364), Nucin (AF005384), and Transferrin (DQ375762). Control: a fifth-instar larva of silkworm; BmRelish1t, a fifth-instar larva of G1 BmRelish1t-induced transgenic silkworm. Relative expression levels are presented as the mean ± SD (n = 3); *p < 0.05 versus control.

Table 1. Identification of the genomic insertion of PG-3xP3EGFP-BmA3BmRelish1t vector into the G1 transgenic silkworm genome by inverse PCR. Insertion length means the length of genomic DNA between the Sau3AI site and the 3′ insert boundaries of the vector. TTAA-duplicated sequences appearing at 3′ insert boundaries are underlined.

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Table 2. Primers used in the real-time PCR. Primer design was based on the GenBank database.

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