柑橘全爪螨对螺虫乙酯关键抗性相关基因筛选及功能验证

Citrus, as the most important fruit in Jiangxi Province, is vulnerable to the citrus red mite Panonychus citri (McGregor) which greatly hinders the development of the citrus industry. Novel tetronic acid insecticide – spirotetramat has enjoyed increasing application to the resistance management of P. citri due to its good controlling efficacy. The previous study found that P. citri had exhibited about 1.5-6 fold cross-resistance to spirotetramat in southern Jiangxi, but its resistance mechanism remains unclear. This project intends to establish a P. citri strain with high level of resistance to spirotetramat by resistance selection in laboratory. RNA-seq and iTRAQ technologies are to be respectively applied to analyzing the differences of gene expression profiles and proteomes between the resistant strain and the susceptible strain, and bioinformatics is to be adopted in studying the functions of differentially expressed genes and differential proteins as well as their relationships with resistance. The association analysis of proteomic dataset and gene expression profiles intends to find the key genes. The functions of the key genes will be studied with the RNAi and gene recombination technologies in order to clarify the role of the key genes. The results of the study will lay the foundations for the molecular mechanisms of resistance to spirotetramate in citrus red mite, and can also shed light on the access to molecular monitoring methods and the establishment of management strategies for field resistance.

柑橘是江西省最重要的水果，而柑橘全爪螨是影响柑橘产业发展的首要害螨。新型季酮酸类杀虫杀螨剂—螺虫乙酯对柑橘全爪螨具有良好的防效，正逐渐应用于其抗性治理。本课题组前期研究发现，赣南地区的柑橘全爪螨已对螺虫乙酯产生了1.5－6倍的抗性，但其抗药性机制尚不清楚。本项目拟采用室内药剂汰选，建立对螺虫乙酯具有高水平抗性的柑橘全爪螨品系；利用RNA-seq和iTRAQ技术分别分析基因表达谱及蛋白质组在抗性品系与敏感品系之间的差异，并借助生物信息学分析差异基因和差异蛋白的功能及其与抗性产生的关系；关联分析iTRAQ蛋白质组数据和RNA-seq表达谱数据，筛选与该抗性相关的关键基因；利用RNAi及基因体外重组技术验证关键基因功能，探明其在抗药性产生中的作用。预期研究结果可为柑橘全爪螨对螺虫乙酯的抗药性分子机理研究奠定基础，同时可为田间抗药性分子监测手段的建立及抗药性治理策略的制定提供思路。

柑橘是江西省最重要的水果，而柑橘全爪螨是影响柑橘产业发展的首要害螨。新型季酮酸类杀虫杀螨剂—螺虫乙酯对柑橘全爪螨具有良好的防效，正逐渐应用于其抗性治理。本课题组前期研究发现，赣南地区的柑橘全爪螨已对螺虫乙酯产生了低水平抗性，但有关该抗药性的相关研究仍比较少。本项目对柑橘全爪螨进行了室内抗药性选育，建立了对螺虫乙酯具有68.2倍抗性的品系；确定了与抗性产生相关的主要解毒酶系—多功能氧化酶，并克隆了6个P450单加氧酶基因，然后通过荧光定量PCR测定了以上基因在抗性品系中表达量的变化；对表达量差异最大的CYP4CL2基因和CYP392B6基因进行了RNAi，并通过毒力试验分析验证了其功能，确定了其与柑橘全爪螨对螺虫乙酯抗药性的产生有关；扩增并比对了柑橘全爪螨抗性品系与敏感品系的ACCase基因序列，但未发现基因突变，而进一步的荧光定量PCR试验发现，抗性品系体内ACCase基因表达量相对于敏感品系上调了3.18倍，此结果表明ACCase基因表达量的上调可能与害虫抗药性的形成有关。此外，本课题研究了柑橘全爪螨对螺虫乙酯的抗性稳定性，结果表明柑橘全爪螨对螺虫乙酯的抗性并不稳定，呈显著衰退趋势；测定了抗性柑橘全爪螨品系对甲氰菊酯、氯氟氰菊酯等10种杀虫剂的交互抗性，筛选出了噻螨酮、唑螨酯等可在田间交替轮换用于柑橘全爪螨防治的农药品种。以上结果可为柑橘全爪螨对螺虫乙酯抗药性治理策略的制定提供思路。