Zhu Y C, Dowdy A K, Baker J E
Grain Marketing and Production Research Center, ARS-USDA, Manhattan, KS 66502, USA.
Insect Biochem Mol Biol. 1999 May;29(5):417-25. doi: 10.1016/s0965-1748(99)00018-1.
Carboxylesterase-like enzyme cDNAs have been cloned and sequenced from malathion-resistant and susceptible strains of the parasitoid Anisopteromalus calandrae (Howard) (Hymenoptera: Pteromalidae). The cDNAs consist of 1963 nucleotides including a 35 bp untranslated 5'-end, a 1596 bp open reading frame, and a 332 bp untranslated 3'-end. The open reading frame encodes 532 amino acid residues. The predicted protein sequence from these cDNAs includes 2 potential N-glycosylation sites, a carboxylesterase type-B serine active site FGGDSENVTIFGESAG, and conserved residues Ser187, Glu317, and His432 to function as the catalytic triad. The predicted carboxylesterase-like enzyme sequence is most similar to that of the carboxylesterase from the peach-potato aphid, Myzus persicae with 45% sequence identity. Alignment of the parasitoid carboxylesterase-like enzyme cDNAs revealed that there are two nucleotide differences in the open reading frame between the parasitoid strains, including a silent mutation and a point mutation that presumably causes a gene product difference. A nucleotide thymine at position 658 in the susceptible strain cDNA is replaced by a guanine in the resistant strain cDNA. This substitution leads to an amino acid change from tryptophan (Trp220) in the susceptible strain to glycine (Gly220) in the resistant strain. This substitution is genetically linked to resistance but it is not known how or if this amino acid substitution affects detoxification of malathion. Northern blot analyses demonstrated that expression level of the carboxylesterase-like enzyme mRNA in adult A. calandrae is approximately 30-fold higher in the resistant strain relative to that in the susceptible strain. Southern analysis indicated that Pst I or Eco RI restriction sites are different in the two strains. Both a modified gene structure and an increase in expression of carboxylesterase may be responsible for the high level of resistance found in this beneficial wasp.
已从寄生蜂米象金小蜂（Howard）（膜翅目：金小蜂科）的马拉硫磷抗性和敏感品系中克隆并测序了类羧酸酯酶的cDNA。这些cDNA由1963个核苷酸组成，包括一个35bp的5'端非翻译区、一个1596bp的开放阅读框和一个332bp的3'端非翻译区。开放阅读框编码532个氨基酸残基。从这些cDNA预测的蛋白质序列包括2个潜在的N-糖基化位点、一个B型羧酸酯酶丝氨酸活性位点FGGDSENVTIFGESAG，以及作为催化三联体发挥作用的保守残基Ser187、Glu317和His432。预测的类羧酸酯酶序列与桃蚜Myzus persicae的羧酸酯酶序列最相似，序列同一性为45%。对寄生蜂类羧酸酯酶cDNA的比对显示，寄生蜂品系之间的开放阅读框存在两个核苷酸差异，包括一个沉默突变和一个可能导致基因产物差异的点突变。敏感品系cDNA中第658位的核苷酸胸腺嘧啶在抗性品系cDNA中被鸟嘌呤取代。这种取代导致氨基酸从敏感品系中的色氨酸（Trp220）变为抗性品系中的甘氨酸（Gly220）。这种取代与抗性在遗传上相关，但尚不清楚这种氨基酸取代如何或是否影响马拉硫磷的解毒作用。Northern印迹分析表明，抗性品系中成年米象金小蜂类羧酸酯酶mRNA的表达水平比敏感品系高约30倍。Southern分析表明，两种品系中Pst I或Eco RI限制性酶切位点不同。羧酸酯酶基因结构的改变和表达的增加可能是这种有益黄蜂中高水平抗性的原因。
