广西四大自然保护区苏云金芽胞杆菌cry基因资源的.doc

研究报告 Research Report 广西大王岭和大明山自然保护区苏云金芽孢杆菌收集与鉴定 谢柳1,2 张文飞1,2 全嘉新1,2 刘卓明1,2 叶大维3 李有志1 方宣钧2* 1广西大学生命科学与技术学院, 南宁, 530005; 2海南海德热带农业资源研究所, 三亚, 572025; 3南开大学生命科学院, 天津, 300071 *通讯作者, xuanjunfang@ 摘 要 从广西大王岭和大明山两个自然保护区共采集到土样264份，共分离出597株芽孢杆菌，通过光学和电子显微镜检观察，16株分离株观察到伴胞晶体蛋白，初步确定为苏云金芽胞杆菌(Bacillus thuringiensis, Bt)，出菌率为6.06%。在16株Bt分离株中，有4株在芽孢形成过程中能产菱形晶体蛋白，其余12株能产圆形和其他形状的晶体蛋白。利用PCR-RFLP方法和SDS方法对16株Bt分离菌进行了蛋白和基因型的鉴定，结果表明，16株分离株中含有4株cry1Ac基因，表达约130 kD的晶体蛋白，其中含有cry30基因和cry40基因的菌株分别1株和3株，表达大约75 kD的晶体蛋白；另外8株Bt菌株表达蛋白大小不一，其基因型尚不能确定，有待进一步分析。生物测定表明，产菱形晶体含有cry1Ac基因的4株Bt分离株对鳞翅目 关键词 苏云金芽孢杆菌, 收集和鉴定, SDS, PCR-RFLP Bacillus thuringiensis Collection and Isolates Identification from Damingshan and Dawangling Natural Reserves in Gua Xie Liu 1,2 Zhang Wenfei 1,2 Quan Jiaxin 1,2 Liu Zhuoming 1,2 Ye Dawei 3 Li Youzhi 1 Fang Xuanjun 2* 1 College of Life Science and Technology, Guangxi University, Nanning, 530005; 2 Haide Institute of Tropical Agricultural Resources, Sanya, 572025; 3 College of Life Sciences, HYPERLINK / Nankai University, Tianjin, 300071 * Corresponding author, xuanjunfang@ Abstract A total of 264 soil samples were collected from Damingshan and Dawangling Natural Reserves in Guangxi Province of southern China. From those 264 soil samples, 597 Bacillus isolates were harvested; and among them, 16 isolates were further identified to be Bacillus thuringiesis using oil lens optical microscope and scanning electronic microscope. The isolation rate of these 264 soil samples is 6.06%. Among the 16 B. thuringiensis isolates, four isolates were observed to produce diamond parasporal crystal proteins and others produce different shapes of crystal proteins including circular and irregular shapes. The selected 16 isolates were analysed using PCR-RFLP and SDSto identify their protein and genotype. The results showed that four B. thuringiensis isolates were identified as cry1Ac-genotype and encoding about 130 kD parasporal crystal protein; three isolates as cry40-genotype and on