MOSQUITOCIDAL TOXINS--SYNERGISM AND CRYSTAL FORMATION

蚊子传播一些世界上最严重的疾病， 包括疟疾、丝虫病、黄热病和脑炎。与 杀虫剂和抗药性蚊子的出现，以及 对化学农药毒性的日益关注，使用 环境无害的生防剂，如芽孢杆菌 苏云金杆菌(Bt)比以往任何时候都更加重要。Bt亚种。以色列人 (BTI)产生灭蚊晶体蛋白，也称为Delta- 内毒素，在孢子形成过程中结晶包涵体内。近期 研究表明，最佳的灭蚊活动需要 三角洲-内毒素之间的协同作用。此外, 最佳的灭蚊活性取决于产生的 这些三角洲内毒素形成结晶包裹体。我们的长期计划 研究目标是明确协同作用的机理 三角洲-烯毒素之间的相互作用及其过程的表征 用于体内蛋白质结晶。在这项建议中，我们的研究将 关注几种三角洲内毒素，包括CyTA和CryIVD，以及 从Bti分离得到了由CryIVD操纵子编码的20 kDa蛋白。这个 以下是这项为期5年的提案的具体目标：1)分离 并对具有活性的20 kDa蛋白进行了鉴定；2)结合能力的测定 20-kDa蛋白与CyTA的特异性；3)确定 20-kDa蛋白在Bt晶体蛋白合成和转录中的作用 降解；4)确定CyTA之间的协同作用机理 和CryIVD；5)确定了CyTA的结晶机理-- 依赖的杀蚊活性；5)测定细胞色素A 结构(S)负责通过现场导向进行协同 诱变。更全面地了解 这些灭蚊蛋白之间的协同作用将 为设计更有效的昆虫Bt菌株提供新的策略 控制力。了解体内蛋白质的作用机制 结晶不仅对提高药效很重要 并扩大Bt生物杀虫剂的目标范围，但也用于 Bt基因作为一种新型基因表达系统的研究进展 以晶体的形式生产蛋白质。

Mosquitoes transmit some of the world's most serious diseases, including malaria, filariasis, yellow fever, and encephalitis. With the emergence of pesticide- and drug-resistant mosquitoes, and the increased concern over chemical pesticide toxicity, the use of environmentally-safe biological control agents, such as Bacillus thuringiensis (Bt) is more important than ever. Bt subsp. israelensis (Bti) produce mosquitocidal crystal proteins, also call delta- endotoxins, within crystalline inclusions during sporulation. Recent studies suggest that optimal mosquitocidal activity requires synergistic interactions between the delta-endotoxins. In addition, optimal mosquitocidal activity is dependent on the production of these delta-endotoxins as crystalline inclusions. Our long term research objectives are to define the mechanism of synergistic interactions between the delta-enotoxins and characterize the process for in vivo proteins crystallization. In this proposal, our studies will focus on several delta-endotoxins, including CytA and CryIVD, and the cryIVD operon-encoded 20-kDa protein insolated from Bti. The following are the specific aims for this 5-year proposal: 1) Isolate and characterize the active 20-kDa protein; 2) Determine the binding specificity between the 20-kDa protein and CytA; 3) Determine the role of the 20-kDa protein in Bt crystal protein synthesis and degradation; 4) Determine the mechanism of synergy between CytA and CryIVD; 5) Determine the mechanism of CytA's crystal- dependent mosquitocidal activity; and 5) Determine the CytA structure(s) responsible for synergism through site-directed mutagenesis. A more comprehensive understanding of the synergistic interaction between these mosquitocidal proteins will provide new strategies to design more effective Bt strains for insect control. Understanding the mechanism of in vivo protein crystallization is important not only for improving the effectiveness and expanding the target range of Bt biopesticides, but also for consideration of Bt as a novel gene expression system for the over- production of proteins as crystals.