Fibroin heavy chain gene replacement with a highly ordered synthetic repeat sequence in Bombyx mori.
Insect biochemistry and molecular biology(2023)
Abstract
The exceptional quality of silkworm silk is attributed to the amino acid sequence of its fibroin heavy chain (Fib-H) protein. The large central domain of Fib-H, which consists of glycine- and alanine-rich crystalline regions interspersed with amorphous motifs of approximately 30 amino acid residues, is considered crucial for fibrilization and determines the properties of the silk fiber. We established a technical platform to modify the Fib-H core region systematically using transcription activator-like effector nuclease-mediated homologous recombination through a somatic and germline gene knockin assay along with PCR-based screening. This efficient knockin system was used to generate a silkworm strain carrying a mutant Fib-H allele, in which the core region was replaced with a highly ordered synthetic repeat sequence of a length comparable with native Fib-H core. Heterozygous knockin mutants produced seemingly normal cocoons, whereas homozygotes did not and exhibited considerable degradation in their posterior silk glands (PSGs). Cross-sectional examination of the PSG lumen and tensile tests conducted on reeled silk threads indicated that the mutant Fib-H, which exhibited reduced stability in the PSG cells and lumen, affected the mechanical properties of the fiber. Thus, sequence manipulation of the Fib-H core domain was identified as a crucial step in successfully creating artificial silk using knockin technology.
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Key words
Gene knockin,Homologous recombination,TALEN,Fibroin heavy chain gene,silkworm
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