小菜蛾对氟虫腈的抗性机理研究
摘要
小菜蛾(Plutella xylostella L．)是世界性分布的重要农业害虫，由于其生殖周期短，繁殖量大，适应性广，并且可以长距离迁飞，在热带和亚热带地区成为十字花科蔬菜的主要害虫。长期以来对小菜蛾的防治严重依赖于化学合成杀虫剂，因此小菜蛾也成为最容易在田间产生抗药性的农业昆虫。现在小菜蛾对各种长期使用的常规杀虫剂甚至新型的杀虫剂和生物杀虫剂都已经产生了严重的抗性，这样就为小菜蛾的综合治理带来了难度。因此，为深入了解小菜蛾对新型的苯并吡唑类杀虫剂的抗性机制，本文从生化和分子角度研究了小菜蛾对氟虫腈的抗性机理。
1 小菜蛾对氟虫腈抗性与交互抗性的研究
从中国深圳坪山镇菜地采集田间小菜蛾，一部分在室内不接触杀虫剂条件下饲养成为PS对照种群，另一部分经20代氟虫腈筛选得到抗性为352倍的PSF品系。与PS对照种群和ROTH标准敏感品系比较，PSF品系对硫丹和狄氏剂存在明显的交互抗性，与阿维菌素和多杀菌素不存在交互抗性。在PSF品系上，PBO和DEF对氟虫腈没有增效作用。
2 小菜蛾对氟虫腈抗性的生化机理研究
采用室内选育的小菜蛾抗氟虫腈品系(PSF)、对照种群(PS)和敏感品系(ROTH)，比较了小菜蛾不同品系多功能氧化酶(MFO)、酯酶(EST)和谷胱甘肽-S-转移酶(GST)的活性。结果表明，抗性品系、未筛选的对照种群和敏感品系的MFO、EST和GST活力均没有明显的差异。小菜蛾对氟虫腈的高水平抗性可能是与靶标不敏感性有关，而与解毒代谢增强没有关系。
3 小菜蛾GABA受体基因克隆与序列分析
根据已经发表的昆虫GABA受体α亚基基因序列，在保守区域设计简并引物扩增GABA受体基因片段。采用PCR和RT-PCR的方法分别从小菜蛾基因组DNA和cDNA中克隆了171bp的GABA受体基因序列(编码57个氨基酸)，氨基酸序列同源性比较显示这个区域高度保守，与多种昆虫GABA受体的同源性都在99％以上。
分别从ROTH品系、PS种群和PSF品系小菜蛾获得的GABA受体基因片段，显示有两种基因Rdl1和Rdl2。Rdl1存在两个等位基因Rdl1a和Rdl1s，分别对应果蝇
小菜蛾对氟虫睛的抗性机理研究
o妞A受体Rdl基因的野生型和突变型，在Rdl 1 a 302位发生AlaGCC一>SerTCC突变。
RdiZ氨基酸序列与Rdl 15相同，但密码子有较大差异。在ROTH品系中发现存在Rdila
与RdlZ两种类型，而在PS种群与PSF品系中三种类型都存在。
我们推测在小菜蛾中至少存在两个Rdl基因位点，一个编码Rdl la/RdllS基因，另
一个编码RdiZ基因。RdlZ基因可能与小菜蛾对氟虫睛的抗性没有关系，而
Rdl la一>Rdlls的突变可能与小菜蛾对氟虫睛的杭性有关。
The diamondback moth, Plutella xylostella L. (Lepidoptera: Yponomeutidae), is one of the most destructive insects of cruciferous plants throughout the world. The diamondback moth is the first crop pest in the world to develop resistance to DDT, and now the diamondback m oth h as develop r esistance t o n early every c lass o f s ynthetic i nsecticides used against it in the field in many countries. It is also the first insect that had developed resistance to the bacterial insecticide Bacillus thuringiensis in the field.
Fipronil is a member of the novel phenyl pyrazole insecticide class that is active at the neuron-inhibitory GABA-gated chloride channel, the same target of cyclodiene insecticides (such as dieldrin, endosulfan). The role of the detoxification enzymes and GAB A receptor Rdl gene involved in fipronil resistance was investigated in the present study.
1. Cross-resistance of fipronil-selected strain of diamondback moth
A field population collected from Pingshan town of Shenzhen in Dec 2002 was divided into two subpopulations. One subpopulation was reared in the laboratory without exposed to any insecticides and named as PS strain. The PSF strain was derived from another subpopulation after 20 generations of continuous selection with fipronil. The PSF strain developed 352-fold resistance to fipronil and showed obvious cross-resistance to two cyclodiene insecticides dieldrin and endosulfan compared with the PS strain. No cross-resistance to avermectin and spinosad was detected in the PSF strain. Neither piperonyl butoxide (PBO, microsomal oxidase inhibitor) nor S, S, S-tributyl phosphorotrithioate (DBF, esterase inhibitor) reduced the level of resistance to fipronil in the PSF strain.
2. Biochemical characterization of fipronil resistance in the diamondback moth.
Detoxication enzyme assays revealed that there were no difference in activities of Glutathione-S-transferases (DCNB, CDNB), general esterase and monooxygenases (ECOD, MCOD, PNOD) among the susceptible Roth, PS and PSF strains. The results suggested that fipronil resistance observed in the PSF strain was most likely attributed to decreased target
site sensitivity.
3. Cloning and sequence analysis of a gene fragment encoding the GAB A receptor (Rdl)
Two Rdl gene fragments Rdll and Rdl2 were cloned from both genomic DNA and cDNA of the diamondback moth by PCR and RT-PCR. The predicted amino acid sequence shares high similarity (>99%) with GABA receptor homologous from various insects.
Gene fragment Rdll has two alleles, Rdlla and Rdl Is. cDNA sequences and amino acids of Rdlla and Rdlls alleles are identical except for an AlaGCC to SerTCC substitution (homologous to Ala302->Ser mutation in Drosophild). Rdl2 has the same amino acids with Rdlls, but their codon usage is different. Only Rdlla and Rdl2 were detected in the susceptible ROTH strain, however the Rdlla -> Rdlls mutation and Rdl2 were found in both PS population and PSF strain. Rdlla -> Rdlls mutation may play a role in the fipronil resistance of P. xylostella.
1 小菜蛾对氟虫腈抗性与交互抗性的研究
从中国深圳坪山镇菜地采集田间小菜蛾，一部分在室内不接触杀虫剂条件下饲养成为PS对照种群，另一部分经20代氟虫腈筛选得到抗性为352倍的PSF品系。与PS对照种群和ROTH标准敏感品系比较，PSF品系对硫丹和狄氏剂存在明显的交互抗性，与阿维菌素和多杀菌素不存在交互抗性。在PSF品系上，PBO和DEF对氟虫腈没有增效作用。
2 小菜蛾对氟虫腈抗性的生化机理研究
采用室内选育的小菜蛾抗氟虫腈品系(PSF)、对照种群(PS)和敏感品系(ROTH)，比较了小菜蛾不同品系多功能氧化酶(MFO)、酯酶(EST)和谷胱甘肽-S-转移酶(GST)的活性。结果表明，抗性品系、未筛选的对照种群和敏感品系的MFO、EST和GST活力均没有明显的差异。小菜蛾对氟虫腈的高水平抗性可能是与靶标不敏感性有关，而与解毒代谢增强没有关系。
3 小菜蛾GABA受体基因克隆与序列分析
根据已经发表的昆虫GABA受体α亚基基因序列，在保守区域设计简并引物扩增GABA受体基因片段。采用PCR和RT-PCR的方法分别从小菜蛾基因组DNA和cDNA中克隆了171bp的GABA受体基因序列(编码57个氨基酸)，氨基酸序列同源性比较显示这个区域高度保守，与多种昆虫GABA受体的同源性都在99％以上。
分别从ROTH品系、PS种群和PSF品系小菜蛾获得的GABA受体基因片段，显示有两种基因Rdl1和Rdl2。Rdl1存在两个等位基因Rdl1a和Rdl1s，分别对应果蝇
小菜蛾对氟虫睛的抗性机理研究
o妞A受体Rdl基因的野生型和突变型，在Rdl 1 a 302位发生AlaGCC一>SerTCC突变。
RdiZ氨基酸序列与Rdl 15相同，但密码子有较大差异。在ROTH品系中发现存在Rdila
与RdlZ两种类型，而在PS种群与PSF品系中三种类型都存在。
我们推测在小菜蛾中至少存在两个Rdl基因位点，一个编码Rdl la/RdllS基因，另
一个编码RdiZ基因。RdlZ基因可能与小菜蛾对氟虫睛的抗性没有关系，而
Rdl la一>Rdlls的突变可能与小菜蛾对氟虫睛的杭性有关。
The diamondback moth, Plutella xylostella L. (Lepidoptera: Yponomeutidae), is one of the most destructive insects of cruciferous plants throughout the world. The diamondback moth is the first crop pest in the world to develop resistance to DDT, and now the diamondback m oth h as develop r esistance t o n early every c lass o f s ynthetic i nsecticides used against it in the field in many countries. It is also the first insect that had developed resistance to the bacterial insecticide Bacillus thuringiensis in the field.
Fipronil is a member of the novel phenyl pyrazole insecticide class that is active at the neuron-inhibitory GABA-gated chloride channel, the same target of cyclodiene insecticides (such as dieldrin, endosulfan). The role of the detoxification enzymes and GAB A receptor Rdl gene involved in fipronil resistance was investigated in the present study.
1. Cross-resistance of fipronil-selected strain of diamondback moth
A field population collected from Pingshan town of Shenzhen in Dec 2002 was divided into two subpopulations. One subpopulation was reared in the laboratory without exposed to any insecticides and named as PS strain. The PSF strain was derived from another subpopulation after 20 generations of continuous selection with fipronil. The PSF strain developed 352-fold resistance to fipronil and showed obvious cross-resistance to two cyclodiene insecticides dieldrin and endosulfan compared with the PS strain. No cross-resistance to avermectin and spinosad was detected in the PSF strain. Neither piperonyl butoxide (PBO, microsomal oxidase inhibitor) nor S, S, S-tributyl phosphorotrithioate (DBF, esterase inhibitor) reduced the level of resistance to fipronil in the PSF strain.
2. Biochemical characterization of fipronil resistance in the diamondback moth.
Detoxication enzyme assays revealed that there were no difference in activities of Glutathione-S-transferases (DCNB, CDNB), general esterase and monooxygenases (ECOD, MCOD, PNOD) among the susceptible Roth, PS and PSF strains. The results suggested that fipronil resistance observed in the PSF strain was most likely attributed to decreased target
site sensitivity.
3. Cloning and sequence analysis of a gene fragment encoding the GAB A receptor (Rdl)
Two Rdl gene fragments Rdll and Rdl2 were cloned from both genomic DNA and cDNA of the diamondback moth by PCR and RT-PCR. The predicted amino acid sequence shares high similarity (>99%) with GABA receptor homologous from various insects.
Gene fragment Rdll has two alleles, Rdlla and Rdl Is. cDNA sequences and amino acids of Rdlla and Rdlls alleles are identical except for an AlaGCC to SerTCC substitution (homologous to Ala302->Ser mutation in Drosophild). Rdl2 has the same amino acids with Rdlls, but their codon usage is different. Only Rdlla and Rdl2 were detected in the susceptible ROTH strain, however the Rdlla -> Rdlls mutation and Rdl2 were found in both PS population and PSF strain. Rdlla -> Rdlls mutation may play a role in the fipronil resistance of P. xylostella.
引文
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