耐药性已成为当前球虫病防治中的主要障碍，形成机制至今不明。前期研究发现柔嫩艾美耳球虫马杜拉霉素耐药株糖酵解途径相关酶的表达量明显不同于地克珠利耐药株和敏感株。糖酵解是马杜拉霉素作用阶段虫体的主要能量来源，推测糖酵解途径相关酶特性改变是球虫马杜拉霉素耐药性产生的主要原因。本项目以不同药物浓度的马杜拉霉素耐药株、田间分离耐药株以及敏感株为研究对象，对已获得的马杜拉霉素耐药株特有的8个糖酵解途径差异表达酶进行耐药关键酶筛选，通过酵母双杂交和Co-IP技术鉴定其效应分子；分析耐药关键酶及效应分子的SNP/Indel；利用CRISPR/Cas9等技术验证耐药关键酶及效应分子与球虫产生耐药性的关系，获得球虫对马杜拉霉素产生耐药的靶基因，解析球虫对马杜拉霉素产生耐药的分子机制。研究结果不仅能为阐明球虫对其他种类抗球虫药产生耐药性的机制提供参考材料，同时也为球虫耐药性快速分子诊断方法的建立提供技术支撑。

Avian coccidiosis is one of the most harmful diseases which caused by protozoan parasites of the genus Eimeria in the poultry industry worldwide, resulting in great economic losses. At present, treatment of coccidiosis mainly relies on drugs which can be divided two cateories: ionophore drugs or synthetic chemicals. A consequence of long-term reliance on anticoccidial drugs has led to drug resistance in Eimeria. Moreover, the drug resistance is very serious and widespread, which has become the main obstacle to the treatment and control of the disease. However, to date, the mechanism of drug resistance in Eimeria is unknown.. Maduramicin，a monovalent glycoside polyether ionophore antibiotic, can inhibit the growth of the most continually occurring Eimeria species in poultry industry. It acts on the asexual stages of Eimeria and interfere with the normal transportation of cation across cell membranes, increasing the osmotic pressure of coccidian cell, which causes the influx of excess water into cell thereby causing cell membrane disruption and cell death. In order to balance the osmotic pressure, the parasite must to use energy to flush out excess ions from its cell. Glycolysis is the major way for the asexual stages of Eimeria to provide energy. We carried out comparative transcriptome analyses of a drug-sensitive strain (DS) and Maduramicin -resistant strain (MRR) or Diclazuril-resistant strain (DZR)of E.tenella by RNA-seqencing. 8 differentially expressed genes encoded key enzymes which involved in the glycolysis pathway of madulamycin-resistant strain were identified based on GO annotation and KEGG enrichment analysis. These enzymes may be very important for the development resistance of the E.tenella to madulamycin. . In this study, we will analyze 8 differentially expressed enzymes in different drug concentrations resistant strains，field isolated resistant strains and sensitive strains. Then we will screen the key enzymes related to the resistance of E.tenella to madulamycin in the glycolysis pathway. The specific substrates of the key enzymes will be captured by yeast two-hybrid technology and Co-IP. The relationships of these key enzymes and substrates which are important for the development of madulamycin resistance of E.tenella will be verified and determined by genome sequencing and gene editing technology. These results will provide important rationale to understand the molecular mechanism of the development of drug resistance in E.tenella and support for the establishment of rapid molecular detection methods of drug-resistance.