棉 花 学 报    Cotton Science    2007，19(2)：98-101

Isolation and Purification of Antifungal Protein from Paenibacillus to Verticillium dahliae
ZHOU Yan-fen^1，2，DU Hong-fang²，YUAN Hong-shui¹，ZHANG Yuan-liang²，ZHU Bao-cheng^1，2*
(1.Collage of Life Science，Agricultural University of Hebei，Baoding 071001；China；2.Collage of Life Science，Hebei University，Baoding 071002，China )

Abstract：Cotton Verticillium wilt，caused by soilborne fungi，Verticillium dahliae，is found worldwide in cultivated soil. These fungi are important pathogens of crop and landscape plants. Verticillium 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 Verticillium 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 Verticillium 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 NaCl from 0 to 1 mol·L^-1 in 0.1 mol·L^-1 phosphate buffer (pH 7.5). A-Ⅲ，one of the fractions，showed the antagonistic activity to Verticillium dahliae. Collected sample of A-Ⅲ 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.
Key Words：cotton Verticillium wilt；Verticillium dahliae；Paenibacillus；antifungal protein；isolation and purification  [Full Text，2935KB]