家蚕合成分泌优异家蚕/蜘蛛复合丝纤维的研究

Spider dragline (major ampullate) silk possesses extraordinary mechanical properties, and has broad application prospect. However, due to their cannibalistic behavior, it is impossible to obtain spider silk fibers on a large scale by farming spiders. In the present work, the piggyBac transposon-derived vectors containing 32-fold typical repetitive units of MaSp1 or 48-fold typical repetitive units of MaSp2 will be constructed, and transgenic silkworm strains harboring these corresponding piggyBac vectors will be generated using silkworm transgenic technology to specifically express the recombinant (re)-MaSp1 or recombinant (re)-MaSp2. Moreover, the endogenous silk gene, fibroin heavy chain (Fib-H), of the silkworm transgenic lineages obtained in our study will be mutated employing the CRISPR/Cas9 genome editing technology to reduce the silk protein expression levels, and the exogenous recombinant spider silk proteins expression levels would be enhanced. The work will provide guidance on how to further design and optimize the structure of re-spider proteins, will obtain more outstanding composite spider/silkworm silk fibers because of harboring larger molecular weight re-spider proteins with high expression levels, and will be enabled for the potentially broad applications of the silkworm silk fiber.

蜘蛛牵引丝(Dragline silk)拥有优越的性能，具有广泛的应用前景。但由于蜘蛛具有同类相食的行为，不能通过人工饲养获得大量的蜘蛛丝纤维，限制了蜘蛛纤维的应用。本项目拟通过构建包含类似于天然黑寡妇蜘蛛牵引丝基因MaSp1大小的32倍的重复序列单元和MaSp2的48倍重复序列单元质粒，采用转基因技术获得家蚕品种。同时，利用CRISPR/Cas9技术突变家蚕品种内源丝素重链基因，降低内源丝素重链的表达量，提高外源蜘蛛丝蛋白的表达水平。通过本项目的研究，能够大量获得牵引丝蛋白分子量接近天然蜘蛛、蛋白含量更高、机械性能更加优异的复合蜘蛛/家蚕丝纤维，乃至获得性能接近蜘蛛牵引丝的复合新型材料。

蜘蛛丝是一种高分子蛋白纤维，具有其他纤维不可比拟的优良特性。本课题.根据黑寡妇蜘蛛（Black Widow Spider, Latrodectus hesperus）牵引丝蛋白设计、合成并成功构建了包含不同重复单元的类MaSp1和MaSp2的系列载体，利用显微注射的方法，将其整合到家蚕的基因组内，经Q-PCR、Western blotting、杂交等方法的检测，证明已成功获得了多个转蜘蛛丝基因家蚕品系。进一步的分析表明：随着外源重组蛋白分子量的增加，复合丝纤维的机械性能逐渐增强。为进一步改良蚕丝质量奠定了坚实的基础。 .综合上述结果，本研究为探索采用家蚕生物反应器高效表达外源重组蜘蛛丝蛋白提高茧丝纤维的性能奠定了理论基础。同时为后续进一步研究高性能的丝纤维提供了重要的线索。项目资助发表SCI论文3篇，协助培养硕士研究生1名，1名在读。项目投入经费22万，支出17.6491万元。各项支出基本与预算相符合。剩余经费4.3509万元，剩余经费计划用于本项目研究后续支出。