Biological Science Research Center, Integrative Science Center of Germplasm Creation in Western China (CHONGQING) Science City, Southwest University, 400715 Chongqing, China; Key Laboratory for Germplasm Creation in Upper Reaches of the Yangtze River, Ministry of Agriculture and Rural Affairs, 400715 Chongqing, China; Engineering Laboratory of Sericultural and Functional Genome and Biotechnology, Development and Reform Commission, 400715 Chongqing, China.
Biological Science Research Center, Integrative Science Center of Germplasm Creation in Western China (CHONGQING) Science City, Southwest University, 400715 Chongqing, China.
Int J Biol Macromol. 2023 Mar 31;232:123371. doi: 10.1016/j.ijbiomac.2023.123371. Epub 2023 Jan 26.
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.
家蚕茧由丝素、丝胶、蛋白酶抑制剂和未知功能的蛋白质组成。在本研究中，我们专注于 fhx-L1（类丝氨酸蛋白酶抑制剂），它是丝氨酸蛋白酶抑制剂 fhx 的同源物。我们在 44 种鳞翅目昆虫中鉴定了 154 个 fhx 家族基因，在 B. mori 中发现了 7 个 fhx-Ls。Fhx-L1 是这些蛋白质中在丝中最丰富的，并且特异性地在丝腺中表达。免疫荧光分析表明，fhx-L1 被分泌到整个丝胶层中，类似于丝胶 1（ser1）。Western blot 显示，fhx-L1 蛋白含有 N-连接的寡糖链。利用 CRISPR/Cas9 介导的基因编辑技术生成了 fhx-L1（fhx-L1）的纯合突变体。fhx-L1 的茧比野生型（WT）的更大、更蓬松，这归因于丝纤维之间的较低粘附力。我们还发现突变型丝中的β-折叠含量低于 WT 丝，这导致 fhx-L1 丝的机械性能进一步恶化。我们的研究揭示了 fhx-L1 作为丝中主要结构成分的性质和功能。然后，我们的研究为深入研究丝蛋白功能提供了潜在的见解。
