杀虫剂选择压胁迫下黄曲条跳甲抗药性的微进化机制

Microevolution, which focuses on the effects of both genes on traits and selection on mutation, is being received high attention in biology. Insect resistance is a particular example of microevolution. Up to date, studies have shown that metabolic resistance in cytochrome P450-mediated activity reveals evolution plasticity to some extent. For instance, with the stress of selection pressure from the same insecticide, different populations descended from insects of the same species evolutionarily select diverse resistant P450s. Nontheless, a large number of scientific problems which relate to this newly found indication haven't been resolved. To be specific, is the selection of resistant P450 isoforms random with the stress of selection pressure from the same insecticide? .Based on the concepts and theories of evolutionary dynamics, a hypothesis has been put forward in this project that one P450 would be selected as a resistant gene only after the development exceeding a certain "express quantity threshold" during the process of resistance. When that P450 cannot suffer from a rising stress of the selection pressure, other P450s will transform into resistant ones in the similar way, which can be visualized as passing batons in a relay race. Applying real-time quantitative PCR and mathematical modeling, the project will target in studying the resistant strain of Phyllotreta Striolata (Fabricius), finding the expression principles of resistant P450 during microevolution, and appraising the authenticity of the "express quantity threshold" theory mentioned above. Not only motivating biologists to analyse the molecular mechanism of microevolution on insect resistance from a brand new angle, the prospective achievements of this project will also bring important guidance on the management of insect resistance.

微进化研究着重于基因对性状的影响，以及选择对基因变异的作用，是当前生物学领域关注的焦点。昆虫的抗药性是一种微进化现象。研究表明昆虫细胞色素P450介导的代谢抗性表现出一定程度的进化可塑性，如同种昆虫不同种群在相同杀虫剂的胁迫下，进化选择出的抗性P450基因有所不同。但是，当前许多相关科学问题还没得到阐明，如抗性P450基因的选择是随机的吗？本项目结合进化动力学理论，大胆提出科学假说：选择压胁迫下，当某个P450基因突破自身的"表达量阈值"时，才会被优先选择为抗性基因；当该P450基因难以化解选择压的加大胁迫时，其他P450基因将以类似的方式也发展为抗性基因，表现出一种"接力棒"模式。项目将以黄曲条跳甲为研究对象，利用荧光定量PCR及数学建模等方法，研究抗药性发展过程中P450基因表达量的增长规律，并判定"表达量阈值"假说的真实性。项目的研究成果将从全新的角度解析昆虫抗药性微进化的分子机制。

黄曲条跳甲Phyllotreta striolata是十字花科蔬菜生产中最难防治的虫害之一，持续增加使用化学农药的剂量和喷洒药剂的频率，导致抗药性不断增强，增加了防治的难度，严重制约着我国十字花科蔬菜的产业发展。黄曲条跳甲P450基因介导代谢的抗药性分子机理研究，以期有助于黄曲条跳甲抗性监测、治理及新农药的研制具有重要意义。前期调查发现，黄曲条跳甲对哒螨灵抗性形成与解毒酶系中的P450基因表达水平有关，但前期研究的仅限于部分P450的荧光定量 PCR 分析。为了进一步解释黄曲条跳甲抗哒螨灵的代谢抗性机理，我们通过成虫浸液法处理，然后采用高通量测序对两个不同浓度处理的虫体进行转录测序，研究发现，1）通过de novo联合装拼共鉴定出131个CYP家族成员，其中CYP2家族 18个， Mitochondrial 家族10个，CYP3家族47个，CYP4家族56个。2）基于转录测序FPKM表达数据结果和荧光定量分析结果显示，随着药剂胁迫压力的增加，几乎所有CYP6BQ cluster成员表达量也随之增加。据此，我们推断哒螨灵上调的CYP6BQ cluster成员基因可能参与抗哒螨灵的代谢抗性。4）为了进一步研究CYP6BQ cluster成员在黄曲条跳甲在不同组织的分布，通过RNA-seq比较了卵巢、中肠、脂肪体、头部表达量，明确了CYP6BQ cluster主要分布于中肠和脂肪体。3）通过荧光定量PCR比较了哒螨灵敏感品系和抗药性品系发现，11个CYP6BQ cluster成员在抗药性品系表达量显著高于敏感品系。综上所述，在基因水平上黄曲条跳甲抗性形成与P450基因的CYP6BQ cluster表达量的调控有关。