{"title":"Fibroin heavy chain gene replacement with a highly ordered synthetic repeat sequence in Bombyx mori","authors":"Yoko Takasu, Nobuto Yamada, Katsura Kojima, Masatoshi Iga, Fumiko Yukuhiro, Tetsuya Iizuka, Taiyo Yoshioka","doi":"10.1016/j.ibmb.2023.104002","DOIUrl":null,"url":null,"abstract":"<div><p>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 <em>Fib-H</em> 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 <em>Fib-H</em> allele, in which the core region was replaced with a highly ordered synthetic repeat sequence of a length comparable with native <em>Fib-H</em> 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.</p></div>","PeriodicalId":330,"journal":{"name":"Insect Biochemistry and Molecular Biology","volume":null,"pages":null},"PeriodicalIF":3.2000,"publicationDate":"2023-08-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Insect Biochemistry and Molecular Biology","FirstCategoryId":"97","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0965174823000966","RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
引用次数: 0
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.
期刊介绍:
This international journal publishes original contributions and mini-reviews in the fields of insect biochemistry and insect molecular biology. Main areas of interest are neurochemistry, hormone and pheromone biochemistry, enzymes and metabolism, hormone action and gene regulation, gene characterization and structure, pharmacology, immunology and cell and tissue culture. Papers on the biochemistry and molecular biology of other groups of arthropods are published if of general interest to the readership. Technique papers will be considered for publication if they significantly advance the field of insect biochemistry and molecular biology in the opinion of the Editors and Editorial Board.