Purification, molecular cloning and heterologous expression of a glutathione S-transferase involved in insecticide resistance from the rice brown planthopper, Nilaparvata lugens.
Purification, molecular cloning and heterologous expression of a glutathione S-transferase involved in insecticide resistance from the rice brown planthopper, Nilaparvata lugens.
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DOI：
10.1042/bj3620329
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发表时间：
2002-03
期刊：
影响因子：
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通讯作者：
J. Vontas;G. Small;Dimitra Nikou;H. Ranson;J. Hemingway
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文献类型：
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作者：
J. Vontas;G. Small;Dimitra Nikou;H. Ranson;J. Hemingway
最近在褐飞虱中发现了一种新的基于谷胱甘肽 S-转移酶 (GST) 的拟除虫菊酯抗性机制 [Vontas, Small 和 Hemingway (2001) Biochem. J. 357, 65-72]。为了确定与赋予这种抗性有关的 GST 的性质，通过阴离子交换和亲和层析对来自抗性和易感褐飞虱菌株的 GST 进行了部分纯化。以前与抗性相关的大部分过氧化物酶活性仅限于与亲和柱结合的部分，在抗性昆虫中该活性显着升高。编码 GST (nlgst1-1) 的 cDNA 克隆是从拟除虫菊酯抗性菌株中分离出来的，这是第一个报道的半翅目 GST 序列，与其他昆虫 I 类 GST 的氨基酸一致性高达 54%。 Northern 分析表明 nlgst1-1 在抗性昆虫中过度表达。 nlgst1-1 在大肠杆菌中表达、纯化和表征。重组蛋白与 S-己基谷胱甘肽亲和基质结合的能力、其底物特异性及其免疫学特性证实该 GST 是来自褐飞虱 GST 升高子集的一种。重组nlgst1-1的过氧化物酶活性表明它通过对拟除虫菊酯诱导的脂质过氧化产物的解毒而具有抗性作用。对抗性和敏感菌株基因组 DNA 的 Southern 分析表明，基于 GST 的杀虫剂抗性可能与褐飞虱的基因扩增有关。
A novel glutathione S-transferase (GST)-based pyrethroid resistance mechanism was recently identified in Nilaparvata lugens [Vontas, Small and Hemingway (2001) Biochem. J. 357, 65-72]. To determine the nature of GSTs involved in conferring this resistance, the GSTs from resistant and susceptible strains of N. lugens were partially purified by anion exchange and affinity chromatography. The majority of peroxidase activity, previously correlated with resistance, was confined to the fraction that bound to the affinity column, which was considerably elevated in the resistant insects. A cDNA clone encoding a GST (nlgst1-1) - the first reported GST sequence from Hemiptera with up to 54% deduced amino-acid identity with other insect class I GSTs - was isolated from a pyrethroid-resistant strain. Northern analysis showed that nlgst1-1 was overexpressed in resistant insects. nlgst1-1 was expressed in Escherichia coli, purified and characterized. The ability of the recombinant protein to bind to the S-hexylglutathione affinity matrix, its substrate specificities and its immunological properties confirmed that this GST was one from the elevated subset of N. lugens GSTs. Peroxidase activity of the recombinant nlgst1-1 indicated that it had a role in resistance, through detoxification of lipid peroxidation products induced by pyrethroids. Southern analysis of genomic DNA from the resistant and susceptible strains indicated that GST-based insecticide resistance may be associated with gene amplification in N. lugens.