Identification and functional study of fhx-L1, a major silk component in Bombyx mori.

The silkworm cocoon was composed of fibroins, sericins, protease inhibitors, and proteins of unknown function. In this study, we focused on fhx-L1 (fibrohexamerin-like1), which was the homolog of fibroin fhx (fibrohexamerin). We identified 154 fhx family genes in 44 Lepidoptera insects, and seven fhx-Ls were found in Bombyx mori. Fhx-L1 was the most abundant of these proteins in silk and was specifically expressed in the silk gland. Immunofluorescence analysis showed that fhx-L1 was secreted into the whole sericin layers, similar to sericin1 (ser1). Western blotting revealed that the fhx-L1 protein contains N-linked oligosaccharide chains. CRISPR/Cas9-mediated gene editing was used to generate a homozygous mutant of fhx-L1 (fhx-L1). The cocoon of fhx-L1 was larger and fluffier than that of the wild-type (WT), which was attributed to the lower adhesion between silk fibers. We also found that the content of β-sheet in the mutant silk was lower than in the WT silk, which resulted in further deterioration of the mechanical properties of the fhx-L1 silk. Our study revealed the properties and function of fhx-L1 as a major structural component in silk. Then, our study provided a potential insight for in-depth study of silk protein function.