Translucent larval integument and flaccid paralysis caused by genome editing in a gene governing molybdenum cofactor biosynthesis in Bombyx mori

Tsuguru Fujii, Kazunori Yamamoto, Yutaka Banno

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Abstract

Translucency of the larval integument in Bombyx mori is caused by a lack of uric acid in the epidermis. Hime'nichi translucent (ohi) is a unique mutation causing intermediate translucency of the larval integument and male-specific flaccid paralysis. To determine the gene associated with the ohi mutation, the ohi locus was mapped to a 400-kb region containing 29 predicted genes. Among the genes in this region, we focused on Bombyx homolog of mammalian Gephyrin (BmGphn), which regulates molybdenum cofactor (MoCo) biosynthesis, because MoCo is indispensable for the activity of xanthine dehydrogenase (XDH), a key enzyme in uric acid biosynthesis. The translucent integument of ohi larvae turned opaque after injection of bovine xanthine oxidase, which is a mammalian equivalent to XDH, indicating that XDH activity is defective in ohi larvae. RT-PCR and sequencing analysis showed that (i) in ohi larvae, expression of the BmGphn gene was repressed in the fat body where uric acid is synthesized, and (ii) there was no amino acid substitution in the ohi mutant allele. Finally, we obtained BmGphn knockout alleles (hereafter denoted as BmGphn Δ ) by using CRISPR/Cas9. The resulting ohi/BmGphn Δ larvae had translucent integuments, demonstrating that BmGphn is the gene responsible for the ohi phenotype. Our results show that repressed expression of BmGphn is a causative factor for the defective MoCo biosynthesis and XDH activity observed in ohi larvae. Interestingly, all male BmGphn Δ homozygotes died before pupation and showed a flaccid paralysis phenotype. The genetic and physiological mechanisms underlying this flaccid paralysis phenotype are also discussed.

Original languageEnglish
Pages (from-to)11-16
Number of pages6
JournalInsect Biochemistry and Molecular Biology
Volume99
DOIs
Publication statusPublished - Aug 1 2018

Fingerprint

Bombyx
Biosynthesis
molybdenum
paralysis
Bombyx mori
integument
Paralysis
Genes
biosynthesis
Xanthine Dehydrogenase
xanthine dehydrogenase
genome
Larva
uric acid
genes
Uric Acid
larvae
phenotype
Phenotype
Clustered Regularly Interspaced Short Palindromic Repeats

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Insect Science

Cite this

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title = "Translucent larval integument and flaccid paralysis caused by genome editing in a gene governing molybdenum cofactor biosynthesis in Bombyx mori",
abstract = "Translucency of the larval integument in Bombyx mori is caused by a lack of uric acid in the epidermis. Hime'nichi translucent (ohi) is a unique mutation causing intermediate translucency of the larval integument and male-specific flaccid paralysis. To determine the gene associated with the ohi mutation, the ohi locus was mapped to a 400-kb region containing 29 predicted genes. Among the genes in this region, we focused on Bombyx homolog of mammalian Gephyrin (BmGphn), which regulates molybdenum cofactor (MoCo) biosynthesis, because MoCo is indispensable for the activity of xanthine dehydrogenase (XDH), a key enzyme in uric acid biosynthesis. The translucent integument of ohi larvae turned opaque after injection of bovine xanthine oxidase, which is a mammalian equivalent to XDH, indicating that XDH activity is defective in ohi larvae. RT-PCR and sequencing analysis showed that (i) in ohi larvae, expression of the BmGphn gene was repressed in the fat body where uric acid is synthesized, and (ii) there was no amino acid substitution in the ohi mutant allele. Finally, we obtained BmGphn knockout alleles (hereafter denoted as BmGphn Δ ) by using CRISPR/Cas9. The resulting ohi/BmGphn Δ larvae had translucent integuments, demonstrating that BmGphn is the gene responsible for the ohi phenotype. Our results show that repressed expression of BmGphn is a causative factor for the defective MoCo biosynthesis and XDH activity observed in ohi larvae. Interestingly, all male BmGphn Δ homozygotes died before pupation and showed a flaccid paralysis phenotype. The genetic and physiological mechanisms underlying this flaccid paralysis phenotype are also discussed.",
author = "Tsuguru Fujii and Kazunori Yamamoto and Yutaka Banno",
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T1 - Translucent larval integument and flaccid paralysis caused by genome editing in a gene governing molybdenum cofactor biosynthesis in Bombyx mori

AU - Fujii, Tsuguru

AU - Yamamoto, Kazunori

AU - Banno, Yutaka

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