小菜蛾酚氧化酶介导的免疫防御与氯虫苯甲酰胺抗性之间的关联机制

Phenoloxidase (PO) is an important enzyme in the development of diamondback moth (DBM), Plutella xylostella, which is involved in the recognition and melanization of foreign organisms during immune defense responses. In our previous studies, we found that the PO activity, the pro-phenoloxidase (PPO) protein content and transcriptional expression of PxPPO genes in chlorantraniliprole resistant (R) strain were significantly increased than that of the susceptible (S) strain, which the increasing reasons need to be further followed. And hence this project will focus on the correlation and its mechanism of phenoloxidase-mediated immune defense and chlorantraniliprole resistance in DBM. The project plan to detect the PO activity, PPO protein content, transcriptional expression of PxPPO genes, and the hemolymph PPO expression of S strains and R strains, also to investigate the immune defense ability of hemolymph in R strain; the project plan to investigate the variation of the PO protein/gene through the treatment of the R strain larvae with PO inhibitor and chlorantraniliprole, which the aim to analyze the correlation of PO-mediated immune defense and chlorantraniliprole resistance in DBM. This project plan to do the quantitative proteomics analysis of iTRAQ and develop the association analysis together with the previous RNA-Seq data of R and S strain, which the aim to find the different related proteins between R and S strain. The expected results of the project could reveal the correlation and its mechanism between PO mediated immune defense and chlorantraniliprole resistance in P. xylostella, which would shed a scientific light for the integrated management of the resistant P. xylostella.

酚氧化酶（PO）在小菜蛾生长发育过程中具有重要的免疫防御功能。前期研究发现，小菜蛾抗氯虫苯甲酰胺品系的PO活性、酚氧化酶原（PPO）蛋白表达和PxPPO基因的mRNA转录表达水平均显著高于敏感品系，发生这些变化的原因值得关注。本项目将紧紧围绕“小菜蛾PO介导的免疫防御与氯虫苯甲酰胺抗性之间的关联关系”这一科学问题开展研究。项目拟进行抗性品系与敏感品系小菜蛾PO蛋白/基因特性和血淋巴免疫防御差异研究，分析PO参与介导的免疫防御与抗药性之间的关系；通过PO抑制剂和氯虫苯甲酰胺相继处理目标试虫，分析PO蛋白/基因特性等变化，进一步理清PO参与介导的免疫防御与抗药性之间的关联关系；通过iTRAQ和前期获得的RNA-Seq数据，找到与抗药性有关的PO差异蛋白或密切相关蛋白。项目研究结果可阐明小菜蛾PO介导的免疫防御与氯虫苯甲酰胺抗性之间的关联关系及其机理，可为抗性小菜蛾的综合治理提供科学依据。

前期研究发现，小菜蛾抗氯虫苯甲酰胺品系的酚氧化酶（PO）活性、酚氧化酶原（PPO）蛋白表达和PxPPO基因的mRNA转录表达水平均显著高于敏感品系，为探讨发生这些变化的原因。本项目围绕“小菜蛾PO介导的免疫防御与氯虫苯甲酰胺抗性之间的关联关系”这一科学问题开展了深入细致地研究。研究中通过测定小菜蛾对氯虫苯甲酰胺、氟苯虫酰胺的抗性，明确了小菜蛾对不同药剂的交互抗性和抗性发展规律；研究了氯虫苯甲酰胺、氟苯虫酰胺抗性品系小菜蛾酚氧化酶（PO）蛋白活性、蛋白含量和PxPPO转录表达量及与抗性之间的关系；通过对3-羟基-4-甲氧基苯甲醛缩氨基硫脲等三种缩氨基硫脲类化合物及氯虫苯甲酰胺分别先后处理的小菜蛾，进行PxPPO基因的转录水平、PPO2蛋白表达量以及PO酶活力的测定，明确了PO参与介导的免疫防御与抗药性间具有的正相关性；并利用RNA-seq和iTRAQ技术进行了小菜蛾抗性品系转录组学和蛋白质组学的研究及分析，比较了小菜蛾不同抗性品系mRNA转录水平的变化，获得了XM_011565890.1和XM_011557520.1等差异基因；利用荧光定量PCR、酶联免疫和UPLC-MS/MS等技术开展了对氟苯虫酰胺抗性品系小菜蛾PxVg、PxVgR、PxMet、PxTre等生殖发育相关基因表达的研究。研究结果基本揭示了酚氧化酶介导的免疫防御与小菜蛾对氯虫苯甲酰胺、氟苯虫酰胺抗性之间的关联机制，为建立以酚氧化酶为靶标的抗药性治理方案提供了新线索。