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 抗药性蚊子的出现， 对化学农药毒性的日益关注， 环境安全的生物防治剂，如芽孢杆菌 苏云金杆菌（Bt）比以往任何时候都更重要。Bt亚种杆菌以色列 (Bti)产生杀蚊晶体蛋白，也称为δ- 内毒素，在孢子形成期间的结晶内含物内。 最近 研究表明，最佳的灭蚊活性需要 δ-内毒素之间的协同相互作用。 此外，本发明还提供了一种方法， 最佳的杀蚊活性取决于 这些δ-内毒素是晶体内含物。 我们的长期 研究目标是明确协同作用的机制， δ-毒素之间的相互作用，并表征该过程 用于体内蛋白质结晶。 在这项建议中，我们的研究将 重点关注几种δ-内毒素，包括CytA和CryIVD，以及 从Bti分离的cryIVD操纵子编码的20-kDa蛋白。 的 以下是这个五年计划的具体目标：1）隔离 并表征活性20-kDa蛋白; 2）测定结合 20-kDa蛋白质和CytA之间的特异性; 3）确定20-kDa蛋白质和CytA之间的特异性。 20-kDa蛋白在Bt晶体蛋白合成中的作用， 4）确定CytA和CytB之间的协同作用机制。 5）确定CytA的结晶机制- 依赖性杀蚊活性;和5）测定CytA 负责通过定点协同作用的结构 诱变 更全面地了解 这些杀蚊蛋白之间协同相互作用将 为设计更有效的Bt菌株提供了新的策略 控制 了解体内蛋白质的作用机制 结晶不仅对于提高有效性很重要， 扩大Bt生物农药的靶标范围， 考虑Bt作为一种新的基因表达系统， 以晶体形式生产蛋白质。

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.