nAChRs Agβ1R81T 突变导致棉蚜对吡虫啉产生抗性的分子机制

Nicotinic acetylcholine receptor (nAChRs) mediated target resistance mechanisms of the insects to imidacloprid are currently a research hotspot at home and abroad. Previous studies indicated that nAChRs β1 subuit are highly conserved at 81 amino acid position, which can directly act with nitro group of the imidacloprid and is a key determinant of imidacloprid binding to nAChRs. Recently we have found that a much higher level of [3H]imidacloprid-specific binding to the susceptible strain than to the resistant strain of Aphis gossypii. Comparison of the nucleotide sequence of nAChRs β1subunit (Agβ1) genes from resistant and susceptible strain revealed a single point mutation in the loop D region of the resistant strain, causing an arginine to threonine substitution (R81T). In order to clarify the influence of Agβ1R81T mutation on the function, activity, and binding site structure of receptor, and the binding ability of nAChRs to imidacloprid, the recombinant hybrid nAChRs will be expressed in heterozygosis, and the co-immunoprecipitation, radioactive ligand binding, bipolar patch clamp, molecular docking study will be adopted. These researches can identify the molecular mechanisms of imidacloprid resistance in A. gossypii. These researches can not only identify the target resistance mechanisms of insects to imidacloprid abundantly, but also provide new ideas and target model for neonicotinoids molecular design, structural transformation, products innovation and resistance management.

基于昆虫烟碱型乙酰胆碱受体(nAChRs)的吡虫啉抗性机制是目前国内外研究的热点。已有研究发现，位于nAChRs亚基上Loop D 区域的81位氨基酸高度保守，能与吡虫啉的硝基直接作用，并影响与吡虫啉的结合。我们研究表明，与敏感品系相比，室内筛选的高抗吡虫啉棉蚜品系膜蛋白与[3H]吡虫啉的结合明显降低，并在抗性品系的Agβ1亚基的Loop D区域发现一个点突变，导致81位的苏氨酸取代精氨酸（R81T）。本研究应用免疫共沉淀、昆虫杂合nAChRs表达、放射性配体结合、膜片钳、分子对接技术，重点揭示Agβ1R81T突变对棉蚜nAChRs功能和活性、结合位点结构及结合吡虫啉的影响，从分子水平深入探究Agβ1R81T突变在棉蚜对吡虫啉产生抗性中的作用。研究结果不仅可进一步揭示昆虫对吡虫啉的抗性机制，还为新烟碱类药剂的分子设计、结构改造、产品创新和抗性治理提供新思路和理论依据。

室内筛选的高抗吡虫啉品系棉蚜nAChRs的β1亚基上发现了一个点突变，导致81位的精氨酸被苏氨酸取代（R81T），但该突变导致昆虫对吡虫啉产生抗性的分子机制尚不明确。本研究以室内筛选的具有突变的棉蚜抗性品系和敏感品系为研究对象，用吡虫啉持续筛选抗性棉蚜品系，测定了其抗性水平和交互抗性，比较了抗性和敏感品系nAChRs各亚基的表达水平；通过免疫共沉淀技术筛选出了棉蚜內源nAChRs亚基组合，然后体外重组构建和表达棉蚜的重组杂合nAChRs，应用电压钳技术比较了含有点突变和不含点突变的的重组杂合棉蚜nAChRs对乙酰胆碱和吡虫啉的结合差异；同源建模构建棉蚜nAChRs的3D模型，通过分子对接比较了野生型和突变型棉蚜nAChRs与吡虫啉的结合能力。具体研究结果如下：. 抗性棉蚜种群继续用吡虫啉连续筛选至75代，抗性倍数增长至72.60倍，且抗性品系对新烟碱类杀虫剂均产生了大于10倍的交互抗性，但抗性和敏感品系棉蚜体内的nAChRs各亚基的表达量差异不大。免疫共沉淀和免疫耗尽反应结果表明棉蚜Agα3和Agα1均可以与Agβ1在体内组成具有功能活性的异源五聚体型nAChRs。重组杂合受体的功能和活性验证及对吡虫啉的结合能力结果表明，棉蚜nAChRs β1亚基81位氨基酸残基由精氨酸突变为苏氨酸后，能引起nAChRs结合吡虫啉的能力下降。. 采用SWISS-MODEl 在线服务器，选择石纹电鳐（Torpedo marmorata）nAChRs蛋白的D链和C链分别作为棉蚜α3和β1两个亚基分子的模板进行同源建模，利用Autodock 4.0软件分别将吡虫啉与Agα3/ Agβ1R81和Agα3/β1T81进行分子对接。结果表明：Agα3/Agβ1R81与吡虫啉结合时能形成两个氢键，距离分别为1.9 Å和2.1Å；而Agα3/Agβ1T81）与吡虫啉结合时只能形成一个氢键，距离长为3.3Å。说明当81位氨基酸为没有突变的精氨酸时，nAChRs与吡虫啉的结合更加紧密，更加牢固。这也为棉蚜nAChRs β1 亚基R81T突变导致棉蚜对吡虫啉的抗性增高提供了证据。所有研究结果阐明了棉蚜nAChRs Agβ1R81T 突变导致棉蚜对吡虫啉产生抗性的分子机制。