海南岛热带雨林区芽孢杆菌收集及Bt菌鉴定  

Collection of Bacillus and Identification of Bacillus thuringensis Isolates from Tropical Rain Forest Reserves of Hainan Island

作　　者：张文飞[1,2] 全嘉新[2] 谢柳[1,2] 王茜[1,2] 易艳桃 丰玫玫 朱麟[3] 王锐萍[3] 方宣钧[1] 

机构地区：[1]海南海德热带农业资源研究所,海南三亚572025 [2]广西大学生命科学与技术学院,广西南宁530005 [3]海南师范大学生命科学院,海南海口571158

出　　处：《基因组学与应用生物学》2009年第2期265-274,共10页Genomics and Applied Biology

基　　金：中国Bt资源收鉴与利用项目;国家863项目(2006AA022189)资助

摘　　要：本研究系统地对海南岛热带雨林自然保护区进行了土壤样品的采集、芽孢杆菌的分离收集和Bt菌株的鉴定。从尖峰岭热带雨林区、五指山热带雨林区、吊罗山热带雨林区、霸王岭热带雨林区总共采集了土壤样品1882份,采用醋酸钠培养基结合高温方法分离出芽孢杆菌3924份,鉴定出Bt分离株158份,Bt菌株的分离率和出菌率分别为4.03%和8.40%。结果分析表明,海南岛热带雨林区芽孢杆菌及Bt菌株分布对环境和生态表现出一定的规律性,一般海拔900m至1400m的Bt菌株含量高、植被覆盖率高,土壤腐殖质含量高的热带沟谷雨林带Bt菌株含量最高。显微观察发现,获得的Bt菌株其伴胞晶体有菱形、球形、方形、椭球形、不定性等多种形状。利用SDS-PAGE方法对获得的Bt分离株进行了伴胞晶体进行分析,发现伴胞晶体的分子量有20kD到150kD不等。进一步利用PCR-RFLP技术对Bt分离株进行了cry基因型的分析,初步发现这些Bt菌株含有cry1、cry3、cry4、cry6、cry30、cry40等基因型。我们还利用鳞翅目昆虫小菜蛾和鞘翅目昆虫椰心叶甲进行部分Bt分离株的生物测定,初步结果显示本研究鉴定出的Bt分离株具有不同的抗虫靶标,对同一靶标昆虫也表现出不同的杀虫活性。整体而言,本研究结果显示出海南岛热带热带雨林自然保护区因其独特的热带地理生境、自然的生物演化系统,使得热带雨林区蕴藏了Bt菌株资源多样化,值得期待挖掘出一些新的菌株和新的基因资源。In present research we conducted the collection of soil samples from tropical rain forest reserves of Hainan island and isolation of Bacillus and identification of Bacillus thuringensis. Totally 1882 soil samples were collected from the tropical rain forest reserves of Jianfenglin, Wuzhishan, Diaoloushan and Bawanglin, 3924 bacillus isolates were harvested based on the approach of selection of sodium acetate culture medium and treatment of high temperature, and 158 Bacillus isolates were further identified as Bacillus thuringensis isolates. The isolating rate and plating efficiency of Bt isolates do reach to 4.03% and 8.40%, respectively. The results preliminary indicated that the trend of distribution of Bacillus and Bt isolates were comply with the environmental and ecological characters, which shows Bt isolates are productive from the soil samples collected at the height above sea level between 900 m to 1400 m and in the flora of tropical ravine rainforest due to the well cover of humus and vegetation. The identified Bt isolates diversely produce the different shapes ofparasporal crystal, which can be defined as bipyramid, globule, cube, ellipse and irregular shapes based on the microscopic observation. The cry geno-types and their crystal protein profiles of identified Bt isolates were carried out by using PCR-RFLP technique and SDS-PAGE method, the results indicated that the tested Bt isolates be affluent in cry gene diversities, which the genotypes of cry1, cry3, cry4, cry6, cry30 and cry40 were confirmed to express parasporal crystal proteins with molecular weight from 20 kD to 150 kD. Toxicity bioassay of these Bt isolates again larvae of Plutella xylostella and Brontispa longissima was carried out and some of them exhibits specific toxicity to their target insects. Gener-ally, it would have a clue that Bt strain be indigenous to tropical rain forest and that the Bt diversities be dependent to geographical and ecological characters. The research initiative also would be expectation for mining the noval Bt

关 键 词：芽孢杆菌 苏云金芽孢杆菌 伴胞晶体蛋白 cry基因型 海南岛热带雨林区 

分 类 号：Q93[生物学—微生物学]