nAChRs突变介导的西花蓟马对多杀菌素抗性机理研究

Nicotinic acetylcholine receptors (nAChRs) in insects are diversified and play a number of essential physiological roles in the central nervous system. The primary target of spinosad appears to be nAChRs but the target site is unclear. The resistance of Frankliniella occidentalis to spinosad differs in different populations and the resistance mechanisms need to be proved. A next-generation RNA sequencing (RNA-Seq) will be performed in the present study to find and identify differentially expressed genes between spinosad-resistant (>10,000-fold) and susceptible strains of F. occidentalis. The full-length of all the subunits genes of nAChRs will be cloned by using RT-PCR and RACE and the allele sequence will be compared. The functional difference in resistant and susceptible strains is to verify by using site mutation, in vitro expression of subunits including stable mutations in Xenopus oocytes, and two electrode voltage clamp techniques to find the resistance related mutations and then to develop a molecular method for early resistance. As for the cholinergic genes with significant difference in expression quantity will be tested by real-time fluorescent quantitative PCR and their roles in resistance is studied by RNA interference technique. The results will make clear the functional site of spinosad in nAChRs, clarify the resistance mechanism of spinosad in F. occidentalis, and then provide evidence for resistance management. In addition, the results will be important for prolonging and protecting the life of spinosad in field application.

昆虫的烟碱型乙酰胆碱受体（nAChRs）极其多样丰富，在神经系统中发挥着极其重要的作用。多杀菌素的主要作用靶标是nAChRs，但具体的作用位点目前尚不明确，西花蓟马对多杀菌素的抗性具有种群差异性，其抗性机理有待探明。本项目利用高通量RNA测序即RNA-Seq技术，对多杀菌素敏感和抗性西花蓟马品系（抗性倍数>10,000倍）进行差异表达分析，鉴定、筛选与抗性有关的差异表达基因；主要应用RT-PCR及RACE技术克隆所有nAChRs亚基基因全长；采用定向突变、爪蟾卵母细胞体外表达和双电极电压钳技术验证敏感和抗性受体亚基的功能差异，找出与抗性相关的突变位点，研发抗性早期分子检测技术；对于表达差异显著的胆碱能系统相关基因进行荧光定量分析，采用RNAi技术研究差异基因在抗性中作用。研究结果将明确多杀菌素类药剂在nAChRs上的功能位点，阐明西花蓟马对多杀菌素的抗性机理，为西花蓟马的抗性治理提供依据。

对多杀菌素敏感和抗性西花蓟马进行了转录组测序，进行了注释和分析。共有130104个转录本，68538个基因，上调基因多富集在binding的分子功能上，下调基因多富集在水解酶类的分子功能上。对抗性相关基因进行了分类和注释，转录组测序的差异表达基因与qRT-PCR验证结果相关性高度一致。在转录组数据基础上，利用RT-PCR和RACE-PCR技术克隆得到9个西花蓟马nAChR亚基基因，包括α1-α7，β1和β2。这9个基因都具有半胱氨酸环配体门控离子通道蛋白家族基因特征。利用荧光定量PCR技术（qPCR）分析了两个西花蓟马种群的四个龄期烟碱型乙酰胆碱受体亚基的相对表达水平，这些亚基在一龄若虫，二龄若虫，蛹和成虫期都有表达。无论在敏感还是抗性种群中，烟碱型乙酰胆碱受体亚基都有相同的表达模式。即一龄若虫和蛹期表达量要高于二龄若虫和成虫期。没有一个亚基在敏抗种群有显著性的表达差异，表明多杀菌素抗性与这八个亚基的表达量无关。通过8代回交，获得了西花蓟马对多杀菌素抗性的近等基因系种群NIL-R，NIL-R与敏感亲本Ivf03的遗传相似度达到了0.9890，且生物测定结果表明NIL-R对多杀菌素的抗性倍数为36,000倍，与抗性亲本Spin-R的抗性水平相当。在室内建立了5个西花蓟马对多杀菌素不同抗性水平的品系，利用RT-PCR和大量测序获得5个种群的nAChR a6亚基的转录本，建立了缺失型转录本比例与对多杀菌素抗性水平之间的关系。结果表明a6亚基缺失型转录本的比例与抗性倍数之间存在线性关系，说明西花蓟马对多杀菌素的抗性水平越高，缺失型转录本所占的比例越多，因此推测nAChR a6亚基的功能性缺失突变是西花蓟马多杀菌素抗性形成的分子机制。用5个不同地区的西花蓟马田间种群对该模型进行验证，所得卡方值为0.2914（χ2=0.2914<χ23, 0.05=7.815），说明田间种群数据与模型预测数据之间无显著差异，该模型同样适用于田间情况，可作为田间多杀菌素抗性监测的分子诊断方法。