棉花黄萎病拮抗蛋白的分离与纯化

Isolation and Purification of Antifungal Protein from Paenibacillus to Verticillium dahliae

从棉花叶组织中分离到的类芽孢杆菌(Paenibacillus)LC105菌株对棉花黄萎病病原菌大丽轮枝菌具有明显的拮抗作用。该菌株发酵液分别用过滤、高压灭菌及有机溶剂处理,初步证明对大丽轮枝菌产生拮抗作用的物质为蛋白质。LC105菌株发酵液经硫酸铵沉淀、DEAE-Sephadex A-50离子交换层析及Sephacryl S-100凝胶层析分离纯化,得到了分子量为27 kDa的抗菌蛋白。

Cotton Verticillium wilt, caused by soilborne fungi, Verticillium dahlia wide in cultivated soil. These fungi are important pathogens of crop and landscape plants. Verticilliurn dahliae, which can pose serious threat to cotton production, is a vascular wilt pathogen that primarily invades xylem as it moves from the root into the stem. Thus, brown, gray or green streaking in xylem of infected branches are also common symptoms. Some antagonistic bacteria to Verticilliurn dahliae have been reported. Purification of antagonistic protein produced by antagonistic bacteria and thereafter cloning the related gene is also an effective strategy. Paenibacillus LC105 was screened from mesophyll tissue of cotton in Baoding, China. It showed strong antagonistic activity to Verticilliurn dahliae. The fermented broth of LC105 was treated by the three following methods： filtrated sterilization with microvoid filter film, heated at 121℃ for 10 min or deposited in the same volume of chloroform, respectively. The result indicated that the component of strain LC105 responsible for antagonistic activity was protein. Fifty to seventy percent ammonium sulfate was added to the fermented broth for fractional precipitation. The suspension of the precipitate was loaded onto the DEAE-Sephadex A-50 column and eluted by the gradient of NaC1 from 0 to 1 tool ·L^-1 in 0. 1 mol ·L^-1 phosphate buffer （pH 7.5）. A-Ⅲ, one of the fractions, showed the antagonistic activity to Verticilliurn dahliae. Collected sample of AⅢ was loaded onto the Sephacryl S-100 column and washed by 0.1 mol ·L^-1 phosphate buffer （pH 7.5）. One eluted fraction （A-Ⅲ-1） showed antagonistic activity to Verticillium dahliae V190, which was shown one single band by SDS-polyacrylamide gel electrophoresis （SDS-PAGE）. The molecular weight of the antagonistic protein was about 27 kDa.