High-throughput screening for novel Bacillus thuringiensis insecticidal proteins revealed evidence that the bacterium exchanges Domain III to enhance its insecticidal activity

IF 1.5 4区 农林科学 Q2 ENTOMOLOGY Journal of Pesticide Science Pub Date : 2024-07-10 DOI:10.1584/jpestics.d24-011
Ruth Cong, Jing-Tong Hou, Takashi Yamamoto
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Abstract

Approximately 3000 Bacillus thuringiensis (Bt) isolates were screened to discover novel three-domain (3D) Cry proteins active against Helicoverpa zea (corn earworm). From 400 active isolates found during the primary screening, Cry1Ac and Cry2A, which are known to be active against H. zea, were removed using multiplex-primer PCR and high-throughput column chromatography. This process reduced the number of active cultures to 48. DNA segments encoding Domain III of these 48 cultures were amplified by PCR and sequenced. Sequencing revealed two novel Cry1B-type Domain IIIs. Further sequencing of the flanking regions of these domains revealed that one was part of Cry1Bj (GenBank: KT952325). However, the other Domain III lacked Domains I and II. Instead, this Domain III was associated with two open reading frames, ORF1 and ORF2. ORF1 was identified as an ATP-binding protein, and ORF2 as an ATPase, suggesting that Bt exchanges Domain III among homologous Cry proteins.

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高通量筛选新型苏云金芽孢杆菌杀虫蛋白的结果表明,苏云金芽孢杆菌通过交换领域 III 来增强其杀虫活性
对大约 3000 个苏云金芽孢杆菌(Bt)分离株进行了筛选,以发现对玉米穗虫(Helicoverpa zea)具有活性的新型三维(3D)Cry 蛋白。利用多重引物 PCR 和高通量柱层析技术,从初筛发现的 400 个活性分离物中剔除了已知对玉米螟有活性的 Cry1Ac 和 Cry2A。这一过程将活性培养物的数量减少到 48 个。通过 PCR 扩增了这 48 个培养物中编码领域 III 的 DNA 片段并进行了测序。测序发现了两个新的 Cry1B 型 Domain III。对这些结构域侧翼区域的进一步测序发现,其中一个是 Cry1Bj 的一部分(GenBank:KT952325)。然而,另一个结构域 III 缺乏结构域 I 和 II。相反,该结构域 III 与两个开放阅读框(ORF1 和 ORF2)有关。ORF1 被鉴定为 ATP 结合蛋白,ORF2 被鉴定为 ATP 酶,这表明 Bt 在同源 Cry 蛋白中交换了领域 III。
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来源期刊
Journal of Pesticide Science
Journal of Pesticide Science 农林科学-昆虫学
CiteScore
4.30
自引率
4.20%
发文量
28
审稿时长
18-36 weeks
期刊介绍: The Journal of Pesticide Science publishes the results of original research regarding the chemistry and biochemistry of pesticides including bio-based materials. It also covers their metabolism, toxicology, environmental fate and formulation.
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