黑龙江凉水自然保护区苏云金芽孢杆菌的收集与鉴定

黑龙江凉水自然保护区是我国现有保存下来的较大片原始红松林基地之一，总面积6394hm^2，森林覆盖率95％以上。本研究从小兴安岭凉水自然保护区采集土样782份，采用醋酸钠培养基结合高温方法筛选土壤中的芽孢杆菌，通过光学显微镜观察鉴定产生伴胞晶体的苏云金芽孢杆菌（Bacillus thuringiensis．Bt）。总计分离得到芽孢杆菌和苏云金芽孢杆菌分别为1735株和33株，Bt菌株的分离率和出菌率分别为1．90％和4．22％。利用SDS-PAGE和PCR—RFLP方法对筛选获得的成分离株进行了杀虫晶体蛋白和基因型分析，结果表明14株产菱形伴胞的＆菌株，SDS-PAGE电泳分析芽孢后期产生分子量大小130kD蛋白带，PCR-RFLP分析初步鉴定为crylAc基因，其它产圆形或其它不定形晶体蛋白的＆分离菌中，芽孢后期主要蛋白大小为20～150kD不等，PCR—RFLP方法鉴定结合PCR片段测序分析这些菌株含有新型cry4、cry39和cry40基因等。本研究是“中国助资源收鉴与利用”项目组成部分之一，凉水自然保护区苏云金芽孢杆菌的收集与鉴定目的是要对整个东北地区成资源分布和多样性作一个初步评估，实验结果表明东北森林地区具有丰富多样的威菌株和杀虫基因资源。

Identification and Collection of Bacillus thuringiensis from Liangshui Natural Reserves in Heilongjiang Province of Northeast China

Liangshui Natural Reserve in Heilongjiang province of northeast China, with a total area of 6 394 hectare, is one of the Korean Pine Bases presently existent in our country. The forest coverage here is more than 95%. In present research, a total of 782 soil samples were collected from this reserve, 1 735 Bacillus isolates were harvested based on the approach of sodium acetate culture medium and treatment with high temperature, and 33 Bacillus isolates were further identified as Bacillus thuringiensis isolates by observation under oil lens optical microscope. The isolating rate and plating efficiency of Bt isolates do reach to 1.90% and 4.22%, respectively. The selected 33 B. thuringiensis isolates were analyzed using methods of SDS-PAGE and PCR-RFLP to identify their proteins and genotypes. The results showed that fourteen B. thuringiensis isolates with diamond parasporal crystal inclusion, were identified as erylAc-genotype encoding about 130 kD parasporal crystal protein. Other produce different shapes of crystal proteins including globule cube ellipse and irregular shapes as well. The cry-genotypes and their crystal protein profiles of identified Bt isolates were carried out by using SDS-PAGE technique and PCR-RFLP method too, the results indicated that the tested Bt isolates be affluent in cry gene diversities, which the genotypes of cry4, cry39 and cry40 were confirmed to express parasporal crystal proteins with molecular weight from 20 kD to 150 kD. This research is an important component of the project of China National Bt Strains Resource Initiative and is beginning to mine Bacillus and B. thuringiensis strain resources of the whole northeast China. We got the result indicate there are rich in Bt strains and the insecticidal proteins genes resource in the forest region of northeast China.