3种外源因子对家蚕围食膜结构及功能的影响

Effects of Three Exogenous Factors on the Structure and Function of Silkworm Peritrophic Matrix

研究3种外源因子(荧光增白剂FB28、凝集素和灭幼脲)对家蚕围食膜结构、通透性和围食膜蛋白的影响.利用Thermo Scientific Quattro环扫电镜观察围食膜表面结构,尤斯灌流室测量围食膜对不同葡聚糖分子(180 D, 5 kD, 40 kD, 110 kD, 2 000 kD)的透过性,利用蛋白质谱检测围食膜蛋白的变化.结果表明:荧光增白剂FB28、凝集素和灭幼脲均对家蚕围食膜结构和功能有一定影响,但围食膜形态、表面结构和通透性改变程度有所不同.凝集素使围食膜中部增厚,48 h时观察到膜内表面有球形的电子致密结构,通透性显著增大;灭幼脲处理后,围食膜多层结构异常,层与层之间变得疏松,皱褶增多,通透性降低;荧光增白剂FB28对围食膜的破坏最为迅速和剧烈,添食4 h时围食膜能透过2 000 kD的葡聚糖分子,6 h后围食膜基本消失.对荧光增白剂FB28处理后的围食膜进行SDS-PAGE电泳检测发现,在2 h和4 h时蛋白条带与对照组相比差异有统计学意义.质谱分析结果表明:处理后的围食膜新增了与免疫(Apolipophorin-Ⅲ, Beta-1, 3-glucanase)、昆虫生殖能力和抗逆能力(Methuselah-like protein 5)和蛋白质结合(60s ribosomal export protein NMD3)相关的12种蛋白.

To study the effects of fluorescent brightener FB28,wheat germ agglutinin,and diflubenzuron on the structure,permeability and protein of the peritrophic matrix(PM)of silkworm Bombyx mori,the environmental scanning electron microscope was used to observe the surface structure of the PM.A Ussing chamber was used to measure the permeability of the PM to dextrans of different molecular weights(180 D,5 kD,40 kD,110 kD,2000 kD).We used protein mass spectrometry to detect the changes of PM proteins.The results show that Fluorescent brightener FB28,wheat germ agglutinin,and diflubenzuron affected the structure and function of the PM of silkworms,but their effects on morphology,surface structure,and permeability of the PM were different.Wheat germ agglutinin thickened the middle part of the PM Spherical electron-dense structures were observed on the inner surface of the matrix,and the permeability was significantly increased at 48 h.After treatment with diflubenzuron,the multilayer structure of PM was abnormal,the layers became loose with increased folds,and the permeability decreased.FB28 was most rapid and severe agent to destroy the PM.The PM was permeable to dextrans of 2000 kD at 4 h after feeding,and PM disappeared at 6 h after feeding.SDS-PAGE electrophoresis showed that there were differences in protein bands between the treatment group and the control group at 2 h and 4 h.The results of mass spectrometry showed that 12 new proteins related to immunity(apolipophorin-III,beta-1,3-glucosamine),insect reproductive capacity and stress resistance(Methuselah like protein 5)and protein binding(60s ribosomal export protein NMD3)presented in the PM after treatment.