Romão Tatiany Patrícia, de Melo Chalegre Karlos Diogo, Key Shana, Ayres Constância Flávia Junqueira, Fontes de Oliveira Cláudia Maria, de-Melo-Neto Osvaldo Pompílio, Silva-Filha Maria Helena Neves Lobo
Department of Entomology, Centro de Pesquisas Aggeu Magalhães/Fundação Oswaldo Cruz, Recife-PE, Brazil.
FEBS J. 2006 Apr;273(7):1556-68. doi: 10.1111/j.1742-4658.2006.05177.x.
The entomopathogen Bacillus sphaericus is an important tool for the vector control of Culex sp., and its effectiveness has been validated in field trials. The appearance of resistance to this bacterium, however, remains a threat to its use, and attempts have been made to understand the resistance mechanisms. Previous work showed that the resistance to B. sphaericus in a Culex quinquefasciatus colony is associated with the absence of the approximately 60-kDa binary toxin receptor in larvae midgut microvilli. Here, the gene encoding the C. quinquefasciatus toxin receptor, Cqm1, was cloned and sequenced from a susceptible colony. The deduced amino-acid sequence confirmed its identity as an alpha-glucosidase, and analysis of the corresponding gene sequence from resistant larvae implicated a 19-nucleotide deletion as the basis for resistance. This deletion changes the ORF and originates a premature stop codon, which prevents the synthesis of the full-length Cqm1. Expression of the truncated protein, however, was not detected when whole larvae extracts were probed with antibodies raised against an N-terminal 45-kDa recombinant fragment of Cqm1. It seems that the premature stop codon directs the mutated cqm1 to the nonsense-mediated decay pathway of mRNA degradation. In-gel assays confirmed that a single alpha-glucosidase protein is missing from the resistant colony. Further in vitro affinity assays showed that the recombinant fragment binds to the toxin, and mapped the binding site to the N-terminus of the receptor.
昆虫病原体球形芽孢杆菌是控制库蚊属媒介的重要工具，其有效性已在田间试验中得到验证。然而，对这种细菌产生抗性的现象仍然威胁着它的使用，人们一直在尝试了解其抗性机制。先前的研究表明，致倦库蚊群体对球形芽孢杆菌的抗性与幼虫中肠微绒毛中约60 kDa二元毒素受体的缺失有关。在此，从一个敏感群体中克隆并测序了编码致倦库蚊毒素受体Cqm1的基因。推导的氨基酸序列证实其为α-葡萄糖苷酶，对抗性幼虫相应基因序列的分析表明，一个19个核苷酸的缺失是抗性的基础。这种缺失改变了开放阅读框并产生了一个提前终止密码子，从而阻止了全长Cqm1的合成。然而，当用针对Cqm1 N端45 kDa重组片段产生的抗体检测整个幼虫提取物时，未检测到截短蛋白的表达。似乎提前终止密码子将突变的cqm1导向了mRNA降解的无义介导衰变途径。凝胶内分析证实抗性群体中缺少一种α-葡萄糖苷酶蛋白。进一步的体外亲和分析表明，重组片段与毒素结合，并将结合位点定位到受体的N端。
