Mechanism of silk secretion revealed by proteomic analysis of silkworm cocoons with fibroin light chain mutations

Abstract

Silkworm is an economically important insect due to its efficient production of silk proteins. Silk itself and the silk trade have enriched human civilization through art and culture and contributed to early globalization in the Silk Road era for nearly two thousand years. Although a large number of studies on silk have been carried out, the mechanism of silk secretion in silkworms has not been thoroughly studied thus far. As the main component of fibroin, fibroin light chain (Fib-L) plays a key role in the secretion of silk. In this study, we constructed a homozygous Fib-L gene mutant population of a nonpractical variety using the CRISPR/Cas9 system. The homozygous mutants displayed a thin cocoon layer, but their viability was not affected by the Fib-L mutation. Furthermore, a comparative proteomic analysis of homozygous mutant cocoons and wild-type cocoons was performed. Strikingly, fibrohexamerin (P25) was secreted almost normally in the homozygous mutant. Further analysis of cocoon proteins revealed that the mutant responded to greater environmental stress caused by a dramatic decrease in fibroin by significantly increasing the secretion of protease inhibitors. These results will further help explain the silk secretion mechanism of silkworm. SIGNIFICANCE: This study generated a homozygous Fib-L gene mutant population of a nonpractical variety using the CRISPR/Cas9 system. The homozygous mutants displayed a thin cocoon layer, but their viability was not affected by the Fib-L mutation. Furthermore, a comparative proteomic analysis of homozygous mutant cocoons and wild-type cocoons was performed. The analysis of the abundance of silk proteins in the cocoons revealed that P25 could be secreted almost normally. The analysis of the abundance of cocoon proteins other than silk proteins showed that the homozygous mutants responded to greater environmental stress by increasing the secretion of defense-related proteins, such as protease inhibitors. These results will further help explain the silk secretion mechanism of silkworm.

Keywords: CRISPR/Cas9; Fibrohexamerin; Fibroin light chain; Protease inhibitors; Silkworm.