应用免疫胶体金银染色技术定位玉米内生细菌

 本文利用光镜免疫胶体金银染色技术对玉米内生细菌进行了定位研究。用分离、筛选于玉米体内具有生防潜力的优势菌枯草芽孢杆菌B20-006菌株的特异性蛋白,制备兔抗血清作为一抗,二抗为金标记羊抗兔IgG,30%甘油-1%戊二醛固定组织,进行冷冻切片免疫胶体金银染色光镜观察。结果表明:枯草芽孢杆菌液浸根处理后5~7d (三叶期)的组织切片中菌体有大量金银颗粒沉淀,内生细菌在根、茎、叶均有分布,大多寄生在植物组织的细胞间隙,菌量根部大于茎和叶部,根、茎、叶的菌量之比为12:3:1,证明枯草芽孢杆菌B20-006菌株为玉米内生细菌,且可在玉米体内繁殖和传导。

Detection of endophytic Bacillus subtilis B20-006 in corn plant with immuno-gold silver staining techniques

Immuno-gold silver staining (IGSS) was used to detect a corn endophytic Gram negative bacterial strain Bacillus subtilis B20-006, a potential biocontrol agent. Polyclonal antisera as primary antibody were raised in a rabbit against the specific protein of B20-006, which was isolated from corn plants. The bacterial cells in roots, stem, and leaves (five or seven days after root treatment with B20-006 at 3-leaf stage seedling) were localized through following procedure:fixing tissues with a mixture of 30% glycerol and 1% glutaraldehyde, freezing slice fraction of corn tissue, incubating using a primary antibody and a gold-labeled (10 nm) secondary antibody (anti-rabbit IgG) conjugate, and enhancing with silver. Mass gold and silver particles detected around the bacteria indicated that the bacteria distributed widely in the intercellular space of root, stem and leaf. The bacteria population was usually much higher in root than in stem and leaf. The ratio for the bacterial distribution in root, stem, and leaf was 12:3:1. The results of present study demonstrated that Bacillus subtilis strain B20-006 functioned as an endophyte in corn.