Molecular cloning and heterologous expression of a glutathione S-transferase involved in insecticide resistance from the diamondback moth, Plutella xylostella
Molecular cloning and heterologous expression of a glutathione S-transferase involved in insecticide resistance from the diamondback moth, Plutella xylostella
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DOI：
10.1016/s0965-1748(98)00049-6
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发表时间：
1998-09-01
影响因子：
3.8
通讯作者：
Sun, CN
中科院分区：
文献类型：
--
作者：
Huang, HS;Hu, NT;Sun, CN
小菜蛾幼虫体内存在4种谷胱甘肽S转移酶(GST，EC 2.5.1.18)同工酶。本工作旨在克隆和异源表达与该虫对某些有机磷杀虫剂产生抗性有关的同工酶DBM GST-3，并研究其在抗性品系中高表达的分子基础。逆转录聚合酶链式反应(RT-PCR)从一株抗甲基对硫磷的MPA菌株的中肠mRNA和与GST-3的N-末端及内部氨基酸序列互补的简并引物中扩增出一个128个碱基的DNA产物。以该扩增产物为探针，筛选出一个长度为809个碱基的克隆，编码216个氨基酸的蛋白质(Mr为24,083，等电点为8.50)。在昆虫中，DBM的GST序列PxGST3与MsGST1的氨基酸序列同源性最高(46.3%)。PxGST3mRNA水平在MPA中显著高于敏感品系，Southern杂交结果表明该GST同工酶的表达增加可能与基因扩增无关。异源表达的具有酶活性的PxGST3具有与从DBM幼虫中纯化的GST-3相似的生化和毒理学特性。这是第一个克隆的在杀虫剂抗性中具有明确作用的GST。(C)1998爱思唯尔科学有限公司。保留所有权利。
Four glutathione S-transferase (GST, EC 2.5.1.18) isozymes have been characterized in the larvae of the diamondback moth (DBM), Plutella xylostella L., a cosmopolitan insect pest of crucifiers. This work aimed at cloning and heterologously expressing the cDNA of DBM GST-3, an isozyme involved in this insect resistance to some organophosphorus insecticides, and studying the molecular basis for its increased expression in the resistant strains. Reverse-transcription polymerase chain reaction (RT-PCR) using midgut mRNA from a methyl parathion resistant MPA strain and degenerate primers complimentary to the N-terminal and internal amino acid sequences of GST-3 generated a 128 bp DNA product. A clone of 809 bp, obtained by screening a midgut cDNA library of MPA strain using this PCR product as probe, encoded a protein of 216 amino acids (calculated Mr 24,083 and pI 8.50). This GST of DBM, PxGST3, shared the highest (46.3%) amino acid sequence identity, among insects, to MsGST1 of Manduca sexta. PxGST3 mRNA level was considerably higher in MPA than in susceptible strains, and Southern blots suggested that gene amplifi cation was probably not involved in the increased expression of this GST isozyme. Enzymatically active PxGST3 expressed heterologously in E. coli exhibited similar biochemical and toxicological properties as GST-3 purified from DBM larvae. It is the first cloned GST with a well-defined role in insecticide resistance. (C) 1998 Elsevier Science Ltd. All rights reserved.