【作者】 李凤梅；

【导师】 李平；

【作者基本信息】 四川农业大学 ， 生物化学与分子生物学， 2003， 硕士

【摘要】 苏云金芽孢杆菌Bacillus thuringiensis是一种重要的杀虫微生物，基于它杀虫效果明显，且对人、畜及害虫天敌极少或完全无毒害作用，不污染环境，保持生态平衡，不容易引起昆虫抗性等特点，开展新型苏云金芽孢杆菌资源的筛选、鉴定、分子生物学、发酵等方面的研究工作十分重要。本研究对四川生态条件下的苏云金芽孢杆菌资源进行调查和研究，为新型杀虫基因克隆及转基因抗虫育种、新型杀虫工程菌株的构建和苏云金芽孢杆菌制剂的开发与应用打下基础。 本研究从四川温江土壤中筛选出近279株苏云金芽孢杆菌菌株，结合对菜青虫、稻苞虫、水稻二化螟等害虫的田间防治试验，筛选出9株对多种害虫具有强烈毒杀作用的苏云金芽孢杆菌菌株，对其进行了分类鉴定，实验室毒力测验，杀虫晶体蛋白分离和纯化以及相关基因的克隆等研究。此外，本研究还就菌株对水稻的促生性等相关生物学特性作了研究。获得了以下研究结果： 1．收集四川温江土壤样本94个，利用醋酸钠-抗生素法分离出芽孢杆菌279株。通过形态观察、生理生化鉴定，筛选出9株苏云金芽孢杆菌：39、D₃、H₁₂、D₁、L₁、2、4-2、4-14、A₁。 2．根据杀虫基因设计通用引物和特异性引物，进行PCR扩增。结果表明，9个菌株均含cryⅠ和cryⅤ基因，L₁、39号菌株含cryⅡ基因；39、4-14、2、A₁、D₃菌株含cryⅢ基因。每个菌株不仅分别含有不同基因类型，而且含有同一基因类型中的不同基因亚型。39、2、4-2、4-14菌株含crylAb和crylAc两种cryⅠ类基因亚型，其余菌株则含crylAb、crylAc和crylC三种cryⅠ类基因亚型。 3．针对菌株的生物学特性进行发酵，取发酵产物对菜青虫、稻苞虫、水稻二化螟、小菜蛾等室内毒力测定。结果表明：菌株对蔬菜菜青虫的防治效果最好，平均防效达90％以上；对稻苞虫防效最高达到85.68％；对水稻二化螟防效最高能达到83.67％。从各菌对害虫的综合防效来看，39、D₃菌株均为高毒力菌株。 4．田间小区试验结果表明：此9个菌株对菜青虫均有较好的防效，喷施发酵液72h后，害虫均达到死亡高峰，校正死亡率均在90％以上，最高达到100％。防治稻苞虫小区实验表明，喷施发酵产物6天后，39号菌株校正死亡率为85.06％，D₃菌株为83.59％。用5株菌株发酵产物喷施水稻防治水稻二化螟，结果均表明水稻的枯心数明显少于未喷施Bt菌的水稻枯心数，综合防效显示，39、D3菌株均为高毒力菌株，与室内检测结果一致。 5.对编号为39菌株进行生物学特性研究，其生长曲线符合细菌典型生长曲线特征。在pH为7、培养温度为35℃时生长量最大，其防效受紫外线影响。 6.通过发酵产物浸种、浸芽试验表明，其中39、A:处理后的水稻根长、茎长、根干重和茎干重明显高于清水和LB处理，并可使根系发达。大田试验结果显示，用不同菌株的不同浓度喷施水稻后，穗实粒数、结实率、千粒重都明显高于对照。 7.对编号为39的菌株的杀虫晶体蛋白进行分离、纯化，经SDS一队GE电泳显示，39号菌株主要含3种杀虫晶体蛋白，分子量分别为50kDa、67kDa、90kDao 8.利用c理IAb基因的特异性引物对39号菌株全基因组进行扩增，得到一条1330饰的基因片段，进行了回收、纯化、连接、转化、测序研究。序列分析发现该片段的核昔酸、氨基酸顺序与GenBank公布的呷IAb基因核昔酸和氨基酸同源性达到99%以上。 9.分别以质粒为模板、PCR产物为探针进行斑点杂交，将cry1Ab、cry1Ac、crylC和crym基因定位于菌株质粒上。更多还原

【Abstract】 Bacillus thuringiensis is an important insecticidal microorganism. It has not yet showed toxicity to people , domestic animals and natural enemies of pests and can keep the ecological balance. It is less likely than chemical pesticides to cause field resistance in target insects. So, to study on the screening, identification, molecular biology and fermentation of Bacillus thuringiensis is of importance. Resource of Bacillus thuringiensis was studied in Sichuan in this paper. The research lays the foundation for transferring gene engineering about anti-pest breeding, the construction of the new engineering strains against pests and the application and production of Bacillus thuringiensis pesticide.Nine Bacillus thuringiensis strains were isolated from Wenjiang in Sichuan by Sodium acetate-antibiotic method. Their classification status in microbiology was deduced by studying the physiological and biochemical character. Each strain is toxic to Pieris rapae, Parnara guttata, Chilo suppressalis et al in vitro and field trial . Insecticidal crystal protein produced from 39 strain was isolated and purified, and the crylAb gene of 39 strain was cloned through PCR method. The genes were located on the plasmids of the strains through dot-blot hybridization. In addition, the potential promoting in rice was studied.The main results are summarized as below:1. Two hundred and seventy-nine Bacillus were isolated from ninety-four soils samples collected from Wenjiang in Sichuan by sodium acetate-antibiotic method. Nine Bacillus thuringiensis 39, D3, Hi2, Dj, LI, 2, 4-2, 4-14 and A\ were obtainded according to their physiological and biochemical character.2. Based on PCR method, two-step multiplex Polymerase Chain Reaction with primers designed by the cry genes sequences were used to detect the cry genes from nine strains of Bacillus thuringiensis . cry I and cryV genes were found in all of them ; LI and 39 strains have cry II genes; 39, 4-14, 2, AI and D3 strains have cryIII. genes. Each strain not only has different cry gene type, but also different cry-subclass. For example, 39, 2 and 4-14 contain crylAb and crylAc genes. The others have crylAb, cryl Ac and crylC genes.3. The bioassays against Pieris rapae, Parnara guttata, Chilo suppressalis et al in vitro showed that Bacillus thuringiensis had the best biocontrol effects to Pieris rapa, and the averageeffect is 90%. The highest mortality to Parnara guttat is 85.68%, and to Chilo suppressalis is 83.67%. The total tests showed that 39 and Da are higher toxin Bacillus thuringiensis.4. The controlling experiments in field showed that the biocontrol effect against pests would change by the influence of external environment. Howevert, the control result was consistent with the result in vitro. The best control effect against Pieris rapae was 100%. 39 and D3 strains against Parnara guttata was 85.06%, 83.67% respectively, and against Chilo suppressalis was excellent.5. Study on the biological characteristics of 39 strain indicated that its growth curve was bacteria typical growth curve, including four phases: lag phase, exponential phase, stationary phase and decline phase. The best cultural condition was: pH7, 35 . The Biocontrol effect could be affected by the ultraviolet ray.6. The growth-promoting effect of Bacillus thuringiensis on dry weight of root and step was tested in vitro. The field trail showed that the treatments of Bacillus thuringiensis made the number of efficient seeds, the weight of 1000 seeds, the rate of seeding, and length of spike increased respectively.7. SDS-PAG electrophoresis indicated the toxic protein of 39 strain was consisted of three protein :90kDa, 60kDa and 46kDa.8. With the cryl Ab gene primer, PCR was performed using DNA from 39 as the template and 1330bp fragment was obtained. The recovered fragment was inserted into T-vector, and then the recombinant DNA was transformed into the competent cells of DH5 .Sequence analysis showed that the fragment contains 1330bp, coding a protein of 443 amino acids. Compa更多还原