BACILLUS THURINGIENSIS PROTOXIN STRUCTURE AND SYNTHESIS

苏云金芽孢杆菌原毒素结构和合成

• 批准号: 3284444
• 负责人: Arthur I. Aronson
• 金额: $ 18.9万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-09-05 至 1994-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3284444
• 关键词: Bacillus cereus; Insecta; SDS polyacrylamide gel electrophoresis; aminoacid transport; bacterial genetics; bacterial proteins; binding proteins; bioassay; chemical binding; electroporation; endotoxins; gene mutation; laboratory rabbit; messenger RNA; molecular cloning; nucleic acid probes; nucleic acid sequence; plasmids; protein biosynthesis; protein sequence; protein structure function; site directed mutagenesis; stainings; toxicant interaction; transcription factor; western blottings

The amino acid sequences of all Bacillus thuringiensis (B.t.) delta- endotoxins deduced from gene sequences share certain conserved regions which very likely reflect a similar mode of action. Inclusions containing the delta-endotoxins (or protoxins) are solubilized in insect larval guts and converted to toxins which bind to specific cell membrane receptors. The toxins then insert into the membrane to form or alter cation pores. The sequenced toxins are active on larvae from different insect orders including Lepidoptera, Coleoptera and Diptera. Among the target insects are those which damage trees and crops as well as vectors for pathogens. In fact, WHO is employing a B.t. isolate in Africa for vector control. Since there is likely to be a common mode of action, one protoxin gene has been selected for extensive mutagenic analysis in order to assess the contribution of various portions of the protoxin molecule to processing, specificity and toxicity. Both site-directed and mutagenic oligonucleotide techniques will be used to alter specific residues or regions of the gene. The mutated genes will be cloned back into B.t. so that pure inclusions may be isolated for bioassays and for in vitro vesicle binding and transport experiments. An understanding of the contribution of various conserved regions to these processes should be helpful in the design of new toxins, for elucidating the mode of action and for understanding the basis for resistance. Most B.t. isolates contain multiple protoxin genes which are expressed to different extents. In general, protoxin genes are on large plasmids so differential expression could be influenced by gene copy number, plasmid stability and the availability of transcription factors. These particular parameters will be examined in at least two isolates employing gene-specific probes to measure protoxin gene content., steady state mRNA levels and the presence or absence of a particular protoxin-encoding plasmid. Understanding the regulation of protoxin synthesis would be important for the evaluation of the efficacy of new isolates, for constructing new strains by mating and/or transformation and for determining the stability of certain genes in a particular isolate.

所有苏云金芽孢杆菌（B.t.）δ- 从基因序列推导出的内毒素共有某些保守区域 这很可能反映了类似的作用模式。 夹杂物 含有δ-内毒素（或原毒素）的昆虫 幼虫的内脏，并转化为毒素，结合到特定的细胞膜 受体。 毒素然后插入细胞膜形成或改变 阳离子孔隙。 测序的毒素对不同种类的幼虫都有活性 昆虫目包括鳞翅目、鞘翅目和双翅目。 中 目标昆虫是那些损害树木和农作物以及病媒的昆虫 病原体。 事实上，世卫组织正在雇用一名B.t.在非洲隔离， 病媒控制 由于可能有一种共同的行动模式， 已选择原毒素基因进行广泛的诱变分析， 为了评估原毒素分子的不同部分的贡献， 加工、特异性和毒性。 站点导向和 将使用诱变寡核苷酸技术来改变特异性 基因的残基或区域。 变异的基因会被克隆回来 转化为B.t.从而可以分离出纯的内含物用于生物测定， 体外囊泡结合和转运实验。 了解 各种保守区域对这些过程的贡献应该 有助于设计新的毒素，阐明 行动和了解抵抗的基础。 大多数细菌分离物含有多个原毒素基因， 表达到不同程度。 一般来说，原毒素基因 因此差异表达可能受到基因拷贝的影响 数量、质粒稳定性和转录因子的可用性。 将在至少两个分离株中检查这些特定参数 使用基因特异性探针测量原毒素基因含量，稳定 状态mRNA水平和特定的 原毒素编码质粒。 了解原毒素的调节 综合对评价新药物的功效很重要， 分离物，用于通过交配和/或转化构建新菌株 并用于确定特定基因在特定环境中的稳定性， 孤立。