基于进化和重测序研究柔嫩艾美耳球虫对地克珠利抗药性的分子机制

地克珠利是目前抗球虫药物中作用浓度最低的一种，广泛用于畜禽球虫病的预防。国内外研究均表明地克珠利抗药性球虫在养鸡场普遍存在，但地克珠利抗球虫作用的靶基因及分子机制仍属未知。我们将建立实验进化体系，在实验条件下对群体大小不同的柔嫩艾美耳球虫施加强度不同的药物压力进行正向选择，获得地克珠利抗药虫株的实验进化群体；同时在田间分离地克珠利抗药虫株的自然进化群体。进一步对这些群体及药物敏感群体进行混池测序，通过群体遗传连锁不平衡原理分析抗药性群体基因组的选择性清除区域，找到靶标候选基因，并基于CRISPR-Cas9介导的基因替换等技术进行基因功能验证，最终解析柔嫩艾美耳球虫对地克珠利产生抗药性的分子机制，为建立抗药性分子检测方法提供技术支持。培养研究生5名，发表国际论文3-5篇。

Diclazuril is an anticoccidial drug with the lowest concentration when applied to prevent coccidiosis and thus is widely used. Recent investigations showed that diclazuril-resistant coccidia are very common in the field. However, it is unknown of the target gene(s) or the molecular mechanisms directly related to diclazuril resistance. In this study, we will establish an experimental evolution system to select diclazuril-resistant strains of Eimeria tenella based on population size of the parasites and the concentration of the drug. We will get the evolved populations with diclazuril resistance. We will sequence pooled samples (pool-seq) of diclazuril-resistant and sensitive strains from both laboratory-evolved and field-isolated populations and then analyze the allele frequency to find out the selective sweep region. The candidate genes derived from the above analysis will be validated by CRISPR-Cas9 based gene replace, overexpression or mutation to reveal the mechanisms controlling drug-resistance. After accomplishing this project, 5 graduate students will have been trained and 3-5 international peer-reviewed articles will have been published.