近年来,内蒙古北部草原上出现了叶甲虫Galerucadauria,它的管理仍然主要依赖于使用传统杀虫剂或具有新作用的化学控制。该研究旨在鉴定G.daurica田间种群中与二酰胺杀虫剂抗性相关的突变位点,为合理选择杀虫剂提供参考,避免对二酰胺类杀虫剂的快速抗性发展。我们克隆了G的ryanodine受体基因(GdRyR)的全长,基于推导的氨基酸序列通过同源建模构建三维模型和跨膜区。在G.daurica个体中检测到两个潜在突变位点(Gly4911Glu和Ile4754Met)和等位基因突变频率。此外,它们与两种二酰胺杀虫剂(chloantraniliprole,cyantraniliprole)使用分子对接方法分别进行分析。通过剪接和组装获得GdRyR的全长cDNA序列(GenBank登录号:OP828593),长度为15,399bp,编码5,133个氨基酸。GdRyR与其他鞘翅目昆虫的氨基酸相似性为86.70%-91.33%,具有典型的结构特征。对50个田间叶甲虫进行的个体抗性等位基因突变频率测试已在两个潜在突变位点Gly4911Glu和Ile4754Met上鉴定出12%和32%的杂合个体,分别。GdRyR的I4754M突变体模型对chloantraniliprole和cyantraniliprole的亲和力与野生型没有显着差异,都有非共价相互作用,如氢键,疏水相互作用和π-阳离子相互作用。然而,G4911E突变体模型显示出与两种二酰胺杀虫剂的亲和力降低和作用模式降低,从而影响兰尼碱受体与二酰胺杀虫剂的结合稳定性。总之,GdRyR中的G4911E突变可能是Dauria对二酰胺类杀虫剂产生抗药性的潜在机制,也是今后进行抗药性风险评估和合理应用二酰胺类杀虫剂防治的重点.此外,本研究可为基于兰尼碱受体结构的杀虫剂设计提供参考。
In recent years, the leaf beetle Galeruca daurica has broken out in the northern grasslands of Inner Mongolia, its management still mainly depends on chemical control using traditional insecticides or with novel action. The study was aim to identify mutation locus associated with resistance to diamide insecticides in field population of G. daurica, to provide a reference for rational selection of insecticides and to avoid the rapid resistance development to diamide insecticides. We cloned the full length of the ryanodine receptor gene of G. daurica (GdRyR), constructed 3D model and transmembrane regions by homologous modeling based on deduced amino acid sequence. Two potential mutation loci (Gly4911Glu and Ile4754Met) and allelic mutation frequencies were detected in individuals of G. daurica. In addition, their binding patterns to two diamide insecticides (chlorantraniliprole, cyantraniliprole) were analyzed separately using a molecular docking method. The full-length cDNA sequence of GdRyR (GenBank accession number: OP828593) was obtained by splicing and assembling, which is 15,399 bp in length and encodes 5,133 amino acids. The amino acid similarity of GdRyR with that of other Coleopteran insects were 86.70%-91.33%, which possessed the typical structural characteristics. An individual resistance allelic mutation frequency test on fifty field leaf beetles has identified 12% and 32% heterozygous individuals at two potential mutation loci Gly4911Glu and Ile4754Met, respectively. The affinity of the I4754M mutant model of GdRyR for chlorantraniliprole and cyantraniliprole was not significantly different from that of the wild type, and all had non-covalent interactions such as hydrogen bonding, hydrophobic interactions and π-cation interactions. However, the G4911E mutant model showed reduced affinity and reduced mode of action with two diamide insecticides, thus affecting the binding stability of the ryanodine receptor to the diamide insecticides. In conclusion, the G4911E mutation in GdRyR may be a potential mechanism for the development of resistance to diamide insecticides on G. daurica and should be a key concern for resistance risk assessment and reasonable applications of diamide insecticides for control in future. Moreover, this study could provide a reference for ryanodine receptor structure-based insecticides design.