朱砂叶螨酯酶基因CCE23可变剪接介导甲氰菊酯抗性机制研究

除点突变和差异表达外，酯酶基因以可变剪接方式参与抗药性还未见报道。申请人发现酯酶基因CCE23在朱砂叶螨敏感品系中存在CCE23-V1和CCE23-V2两种变体，但在甲氰菊酯汰选后的抗性亚系中仅剩一种变体CCE23-V2，暗示出可变剪接也是酯酶基因介导抗药性的一种方式，但具体机制尚不清楚。本项目将通过RNAi和转基因果蝇技术鉴定两变体基因的功能与甲氰菊酯抗药性的关系，再采用分子对接技术分析两变体蛋白与甲氰菊酯结合位点的异同，最后通过靶点稳定性检测技术（DARTS）分析甲氰菊酯与两变体蛋白结合稳定性的差异，以及借助气质联用仪分析两变体蛋白对甲氰菊酯的代谢能力及产物结构差异，从基因-蛋白-表型三个层次深入研究CCE23剪接变体与朱砂叶螨甲氰菊酯抗性之间的关系。本项目有望提出并证明一条酯酶基因以可变剪接方式参与抗药性的新途径，为深入理解节肢动物抗药性机制的多样性和复杂性提供理论依据。

Besides point mutation and altered gene expression, it remains unrecorded that carboxyl/cholinesterases (CCEs) have contributed pesticides resistance via alternative splicing. We found two variants (CCE23-V1 and CCE23-V2) of CCE23 gene in susceptible Tetranychus cinnabarinus, but one variant (CCE23-V2) in fenpropathrin resistant strain. The curiosity is how alternative splicing contributes CCE23 to fenpropathrin resistance. To underpin the molecular mechanism, this project plans to firstly investigate the functional role of two variants in fenpropathrin resistance by RNAi and transgenic fly, and then analyze the possible binding site of fenpropathrin in two alternative proteins separately via molecular docking. We will also measure the binding affinity of two alternative proteins towards fenpropathrin independently by drug affinity responsive target stability (DARTS), compare the metabolizing capability of two variant proteins by gas chromatography, and elucidate the structure of metabolites by gas chromatography/tandem mass spectrometry assay (GC-MS/MS). This project will investigate the relationship between alternative variants of CCE23 and fenpropathrin resistance from genotype to phenotype to underpin a new strategy, alternative splicing, of esterase conferring fenpropathrin resistance, which might benefit for deep understanding the diversity and complexity of resistance mechanisms.