GABA量变导致朱砂叶螨对阿维菌素抗性机制研究

Our previous research found that compared to the susceptible strain (SS), the avermectin-resistant strain (AR) of Tetranychus cinnabarinus showed significantly higher intrinsic GABA level, however the underlying mechanism is not clear. The cellular GABA level reflects a dynamic balance between synthesis and catabolism, which determined respectively by the glutamate decarboxylase (GAD) and GABA transaminase (GABA-T); furthermore, physiological GABA level could be fed back and regulate the GABA accumulation through inhibiting the activity of GAD, which were well documented in other animals. In this study, firstly, the GAD and GABA-T would be isolated from SS and AR, respectively, and their activity and kinetics would also be compared. Secondly, the cDNA full length of differently expressed genes such as GAD, GABA-T and GABA receptor, which is related to GABAergic system would be cloned with RNA-Seq (Quantification), RT-PCR and RACE methods, changes of the mRNA expression of these genes in different strains, and with/without avermectin treated would be analyzed using real-time qPCR method. Thirdly, the GAD, GABA-T and GABA receptor genes would be expressed in constructed E. coli or other vectors, the recombinant proteins' enzymatic characteristics and capability of catalyzing the synthesis and catabolism for GABA would be confirmed as well as the ion channel's properties. Fourthly, the cDNA sequences of these three genes would be compared between the SS and AR strains for identifying the possible point mutation related to AVM resistance. Finally, the information about increase of GABA level, dynamic changes of enzymatic activities for GAD and GABA-T, expression changes of GABA receptor and the AVM resistance would be integrated and their correlativities would be analyzed. The new mechanisms about higher GABA level resulted in AVM resistance in T. cinnabarinus would be clarified from physiological, biochemical and molecular levels. The results would raise and elucidate a novel mechanism about AVM-resistance in T. cinnabarinus theoretically, perfect and establish the molecular markers for AVM-resistance detection from practice, also provide new ideas possibly for design special insecticide and acaricide with targets were the components of GABAergic system, which would promote the ability for pesticide creation from molecular design and headstream innovation.

我们研究发现GABA升高可以导致朱砂叶螨对阿维菌素（AVM）产生抗性，但相关的机制却不清楚。本项目拟通过分析谷氨酸脱羧酶（GAD）和GABA转氨酶（GABA-T）活性动态在敏感、抗性中的差异以及这种差异与GABA变化的相关性，明确GABA升高的生理生化机制；RNA-Seq结合RACE技术获得与GABA系统有关的差异表达基因cDNA全长，实时定量PCR技术解析受AVM胁迫前后相关基因mRNA在不同品系中的表达变化，异源表达GAD及GABA受体等基因并进行功能验证，阐明GABA升高导致对AVM抗性的分子机理。研究结果在理论上可能提出并阐明一种朱砂叶螨对AVM抗性的新机制，在实践上不仅可完善并建立AVM抗性检测分子标记，还可能为以节肢动物神经GABA能系统成分（GAD、GABA-T和GABA受体等）为作用靶标设计特定的杀虫、杀螨剂提供新的思路，促进我国农药创制分子设计和源头创新的能力和水平。

阿维菌素类药剂（AVM）是一种具有良好活性的杀虫、杀螨和杀线虫剂，目前被广泛用于农业害虫以及卫生害虫的防治。本项目以阐明朱砂叶螨阿维菌素抗性品系中GABA含量上升机理，研究GABA含量上升对朱砂叶螨化学及生物防治效果的影响为目的，取得了以下几方面的研究结果：. 酶活性测定结果表明阿维菌素抗性品系（AbR）中GABA-T活性显著低于敏感品系（SS），而GAD活性无显著差异。荧光定量PCR（qPCR）结果显示2条GAD基因的表达在SS和AbR品系中无显著差异，而GABA-T基因在AbR品系中显著下调。以上基因表达研究结果与已有的酶活测定结果一致，表明朱砂叶螨AbR品系中GABA含量高于SS品系的分子机制是GABA-T基因表达下调。. 氨己烯酸（Vigabatrin）在活体条件下可以抑制GABA-T的活性，高效液相色谱测定结果显示Vigabatrin处理后4h GABA含量显著升高，8h时降低；而AVM处理后4h、8h朱砂叶螨GABA含量均显著高于对照，表明AVM可以刺激朱砂叶螨GABA的释放，且作用时间长于Vigabatrin。毒力测定结果显示经Vigabatrin预处理后敏感个体对AVM的耐药性提高4.1倍，进一步说明高含量的GABA可以降低AVM的杀螨效果。. 通过饲喂GABA-T基因dsRNA的RNAi法提高朱砂叶螨内源GABA含量，结果显示，饲喂GABA-T基因 dsRNA后其mRNA表达下调33%，GABA含量显著增加，朱砂叶螨爬行速度显著减慢。用阿维菌素LC30、LC50分别处理取食GABA-T-dsRNA的敏感个体，死亡率分别减少12%和18%（与对照相比）。说明通过RNAi的方法提高朱砂叶螨GABA含量后，增加了朱砂叶螨对AVM的忍耐力。. 利用喷施GABA溶液的方法外源增加朱砂叶螨GABA含量后，朱砂叶螨在一定的浓度和时间范围内死亡率下降，对AVM的忍耐力显著增加。. GABA是一种抑制性神经递质，GABA含量上升导致叶螨的爬行速度变慢，进一步的研究中发现GABA含量会导致叶螨更容易被捕食螨捕获。这些研究结果同样也在朱砂叶螨野外品系中被验证。通过该项目的研究，证明了GABA可以调节化学防治和生物防治的效果。