【作者】 陆秀君；

【导师】 李国勋； 杜克久；

【作者基本信息】 河北农业大学 ， 植物病理学， 2005， 博士

【摘要】 苏云金杆菌（Bacillus thuringiensis,Bt）营养期杀虫蛋白（Vegetative InsecticidalProteins,VIPs）具有对鳞翅目昆虫广谱的杀虫活性,且与Bt杀虫晶体蛋白（insecticidal crystal proteins,ICPs）作用机理完全不同。因而,分离克隆新型高毒力的营养期杀虫蛋白（vegetative insecticidal proteins,VIPs）基因对于构建高效广谱的基因工程微生物和培育转基因抗虫植物、控制害虫抗性发展都具有重要的意义。本研究以本实验室特有的Bt菌为材料,利用生物测定和分子鉴定相结合的方法,得到了营养期和孢晶分离期活性较高的Bt LS1和LS8菌株,并系统地开展了两菌株的分子生物学研究。结果如下: 本研究对采自河北省境内的614份土样和自然病死虫进行了苏云金杆菌的分离鉴定,得到苏云金杆菌菌株38株,平均分出率为6.2%。对99株Bt野生株进行了生物活性测定,得到了营养期和孢晶分离期对棉铃虫活性较高的菌株LS1和LS8。菌株LS1、LS8和HD-1对二龄棉铃虫的LC₅₀值分别为4.9×10⁶ cells/mL、3.3×10⁶cells/mL和2.8×10⁷cells/mL,两菌株的活性均高于HD-1菌株。对甜菜夜蛾均活性较差,LS8菌株对甜菜夜蛾的LC₅₀值为6.5×10⁸cells/mL,活性与HD1相当;高毒力LS1和LS8菌株在培养特性上与标准菌株HD-1基本相同。营养期培养物对初孵棉铃虫有很好的毒杀作用,对二龄棉铃虫生长有较好的抑制作用,其中LS8菌株上清液处理对初孵和二龄幼虫的死亡率和体重抑制率分别达到50.3%±3.7%和50.4%±2.4%,LS1菌株对二龄幼虫的体重抑制率达到78.7%±6.6%;对甜菜夜蛾未表现杀虫活性;LS8菌株胞内活性物质对甜菜夜蛾有很强的抑制作用,体重抑制率分别达到45.13%和43.20%。热处理试验表明,营养期培养物中活性成分具有热不稳定性。 对99株Bt野生菌株进行vip3A基因鉴定,并进行了基因定位研究。在55株中扩增得到了1.2kb大小的PCR产物,约有55.6%的菌株含有vip3A基因。其中LS1和LS8菌株引物扩增实验中均检测到了靶片断。生物测定和分子鉴定结果说明两菌株中营养期杀虫活性物质为营养期杀虫蛋白。 对LS1和LS8菌株中的营养期杀虫蛋白基因进行了初步定位研究。结果表明,以质粒DNA为模板可扩增得到约2.3kb片段,而基因组DNA均未扩增得到目的片断,对LS1和LS8菌株的目的片断成功克隆（第三章）,证实为营养期杀虫蛋白基因。初步推断两菌株营养期杀虫蛋白基因可能存在于环形质粒上,而并不存在于染色体DNA上。 克隆了LS1和LS8菌株中的营养期杀虫蛋白基因。利用碱裂解法提取质粒为模板,更多还原

【Abstract】 Bacillus thuringiensis （Bt） is a spore-forming gram-positive bacterium. During sporulation, the intracellular insecticidal crystal proteins （Cry proteins） are produced as phase-bright inclusion. These proteins are toxic to insect larvae in the orders Lepidopetera, Duptera and Coleopteoptera. The Cry proteins from B. thuringiensis have been developed as one of the most successful biological agents in agriculture to control insect pests. The vegetative insecticidal proteins（VIPs） secreted outside the cells during mid-logarithmic growth were found recently. Two novel VIPs which are toxic to Helicoverpa armigera were identified from Bt-LS1 and Bt-LS8 isolates, respectively. The characteristics of Bt-LSl and Bt-LS8, the cloning and expression of VIP3A were reported in this study.Insecticidal activity of the isolates from Bt-LS1 and Bt-LS8 was assayed against H. armigera larvae and Bt var kurstaki strain （HD-1） was used as positive control in this experiment. The result from Cry proteins showed that LC50 of Bt-LS 1 and Bt-LS8 is 4.9 ×10⁶ and 3.3 ×10⁶ cells/mL respectively, which was strong for toxic to H.armigera larvae than HD-1 with LC₅₀ of 2.8 ×10⁷ cells/mL. The result from VIP showed that both Bt-LS1 and Bt-LS8 were highly toxic to H.armigera larvae with a mortality of 50.4%.In this study, screening of Bt strains containing vip3A genes and analysis of gene conservation were reported. vip3A genes of Bt isolates stored in our lab were identified using PCR amplification. The results showed that among 99 isolates, 55 contained this gene. On the basis of identification result, two isolates, Bt LS1 and Bt LS8 , highly toxic to Lepidoptera pests, were chosen and studied on location of vip3A. Results of location showed that plasmid DNA existing vip3A gene in LS1 and LS8 isolate, which vip3A gene successfully cloned in chapter Ⅲ different from other authors’ identified on linear plasmids DNA.Two isolates, which were highly toxic to Lepidoptera pests, Bt-LS1 and Bt-LS8更多还原