黑寡妇蜘蛛牵引丝单一多聚序列对蚕丝性能改良的研究

Improvement of Mechanical Properties of Bombyx mori Silk Based on Type1 Sequence of MaSp1 from Latrodectus hesperus

蚕丝性能的优劣决定了蚕丝的价值和使用领域。本研究人工设计和构建了包含蜘蛛丝MaSp1基因中的32倍type1序列(32×MT1)和红色荧光蛋白表达框的piggyBac转基因质粒pBac[DsRed-32×MT1],通过显微注射家蚕品种兰10蚕卵获得了能够稳定遗传的转基因家蚕品系pBac-32×MT1。经荧光显微镜筛选、插入位点分析、mRNA表达丰度检测以及茧壳蛋白检测,证明了转基因家蚕能够表达融合了蛛蛛丝蛋白和轻链蛋白的重组丝。扫描电镜观察显示,转基因家蚕茧壳和丝纤维表面的形态和结构与野生型蚕品种兰10相近。傅里叶转换红外光谱去卷积分析表明,转基因家蚕丝蛋白二级结构中与丝应力有关的β折叠含量达到63.6%,而野生型品种兰10丝蛋白的β折叠含量约为20%。蚕丝机械性能测定表明,转基因家蚕蚕丝的应力、应变和韧性比野生型家蚕丝分别提升了27.7%、10.7%和45.2%。上述结果表明,在家蚕基因组中导入MaSp1基因的type1单一多聚序列能显著增强重组丝的综合力学性能。

The performance of silk determines the value and application fields of silk.In this study,a piggyBac transgenic plasmid pBac[DsRed-32×MT1]containing 32×type1 sequence of spider silk MaSp1 gene(32×MT1)and red fluorescent protein expression frame was artificially designed and constructed.The piggyBac transgenic vector was microinjected into Lan 10 silkworm eggs to obtain genetically modified silkworm strains with stable inheritance(pBac-32×MT1).The transgenic silkworm was proved to be able to express recombinant silk fused with spider silk protein and light chain protein by fluorescence microscopy screening,insertion site analysis,detection of mRNA expression and detection of protein in cocoon shell.Scanning electron microscope analysis showed that the surface morphology and structure of cocoon shell and silk fiber of transgenic silkworm were similar to those of the wild type Lan 10.Results of the Fourier transform infrared spectroscopy deconvolution analysis showed thatβ-sheet content related to silk stress accounted for 63.6%of the transgenic silkworm,whileβ-sheet content in silk of Lan 10 is approximately 20%.The measurement of mechanical properties of silk showed that the stress,strain and toughness of the transgenic silk increased by 27.7%,10.7%and 44.4%respectively,compared with those of Lan 10.The results showed that the integration of the type1 single polymer sequence of the MaSp1 gene in the silkworm genome can significantly enhance the comprehensive mechanical properties of the recombinant silk.