细胞色素P450酶系介导的黄毛鼠杀鼠灵生理抗性的代谢和分子基础分析

Rattus losea is mainly rodents in the farmland of southern China, seriously endangering food security and human health. The long-term use of anticoagulant rodenticide causes the resistance gradually increasing and the control declining, therefore, the study of resistance mechanism has become an important countermeasure against the resistance of rodents. Currently, the research is mainly focused on mutations of the VKORC1, but this is only one of the resistance sources. Cytochrome P450 metabolism as the important enzyme of rodents which could metabolize rodenticides, can reduce the harm of rodenticide to rodents. This can be a starting point to explain another possible generation of resistance. Lethal feeding period test was conducted to study the resistance status of Rattus losea and screen resistant strains. The project will detect Cyt-P450 activity of susceptible and resistant strains respectively, followed by cloning, sequencing and identifing the corresponding gene, then analysis the differences between two strains, and finally analyze the relationship between these physiological, genetic differences and resistance of Rattus losea resistance. The project will analyze the physiological mechanisms of resistance, improve the resistance mechanism of Rattus losea, and lay the foundation for molecular detection of resistant rodents and appropriate strategies of reducing resistance, propose targeted rodent control measures to reduce the rodent hazards of food security and human health.

黄毛鼠是华南地区农田主要害鼠，严重危害粮食安全和人体健康。抗凝血杀鼠剂的长期使用致使害鼠抗性逐年提高，防治效果不断下降，因此，抗性机制的研究成为应对害鼠抗性的重要对策。目前，抗性机制研究主要聚焦于VKORC1基因的突变，但这只是抗性产生的一种方式。细胞色素P450酶系作为害鼠代谢杀鼠剂的重要酶系，可降低杀鼠剂对害鼠的危害，以此为切入点可阐释抗性产生的另一种可能。本项目采用致死期饲喂法研究黄毛鼠对杀鼠灵的抗性现状，并筛选出抗性品系；分别检测敏感品系和抗性品系Cyt-P450的活性，克隆Cyt-P450抗性相关基因并测序和初步鉴定，分析二者差异，最后分析这些生理和遗传差异与黄毛鼠抗性之间的相互关系，阐释产生抗性的生理机制，完善黄毛鼠抗性的机制分析，为开展害鼠抗性的分子检测以及制定相应的抗性应对策略奠定基础，提出有针对性的鼠害防治措施，降低害鼠对粮食安全和人民身体健康的危害。

黄毛鼠是华南地区农田主要害鼠，严重危害粮食安全和人民生命安全。抗凝血杀鼠剂的长期使用致使害鼠抗性逐年提高，防治效果不断下降，因此，抗性机制的研究成为应对害鼠抗性的重要对策。本项目采用致死期饲喂法研究明确了广东省江门市黄毛鼠对第一代抗凝血杀鼠剂的抗性现状，获得了黄毛鼠的敏感品系和抗性品系；分别检测了抗性品系与敏感品系抗性相关酶的含量和活性，发现黄毛鼠的抗性与血清及肝脏中CYP450的含量和活性没有显著关联，但与肝脏中NQO1的含量和活性、肝脏中Cytb5的含量相关，且敏感鼠高于抗性鼠；克隆和鉴定了抗性相关基因CYP1a1基因和VKORC1基因，分析比较了黄毛鼠个体之间抗性相关基因的差异及其与抗性的关系，获得黄毛鼠CYP1a1基因全长6243bp，包含7个外显子和6个内含子，mRNA全长2623bp，CDS全长1575bp，编码524个氨基酸；VKORC1基因全长2399bp，共筛选到12个变异位点，其中1个为插入缺失位点，外显子1和外显子3各存在1个变异位点，内含子1和内含子2分别存在6个和4个变异位点，外显子1的突变导致第58位氨基酸变异（Arg58Gly），外显子3的突变为同义突变（Cys96Cys），相关性分析显示，黄毛鼠的抗药性与VKORC1基因中SNP位点的相关性不显著, 黄毛鼠抗性的遗传机制可能与其它因素有关。研究结果进一步阐明了黄毛鼠抗性的生理机制，为开展害鼠抗性的分子检测以及制定相应的抗性应对策略奠定基础。