内蒙古地区绵羊主要消化道线虫耐丙硫咪唑分子调控机制研究

Digestive tract nematode disease is the main parasitic disease that affects the healthy development of sheep industry. It has the characteristics of wide distribution, high harm and high infection rate. At present, the main method to control sheep digestive tract nematode disease is the use of chemical anthelmints. Some classic anthelmintic drugs (such as thiadiazole) have been abused for a long period in many areas, thus resistant strains were produced, which severely restricted the deworming Work effectively. Based on the previous investigation, the project will conduct to investigate the albendazole resistance of the main digestive tract nematocide in Inner Mongolia, and the RNA sequencing, including mRNA, miRNA and lncRNA will be done. The bio-informatics method will be used to screen the possible genes, miRNAs and lncRNA related to albendazole resistance. Then the regulation relations among them will be predicted. Technologies such as qPCR, RNAi, overexpression(constructing nematode-specific expression vector) and CRISPR / Cas9 will be used to study the resistance gene and their regulatory sequence, Simultaneously, the function and molecular regulatory mechanisms of the key genes about albendazole resistance will be explored. Finally, the rapid detection method of albendazole resistance will be established. The results lay the foundation for the research on the drug-resistance mechanisms of parasitic nematode in sheep, new drug target screening and new veterinary drug development.

消化道线虫病是影响养羊业健康发展的主要寄生虫病，具有分布广、危害大、感染率高的特点。目前，主要利用化学药物驱虫来防治羊消化道线虫病，一些沿用多年的经典驱虫药（如丙硫咪唑）由于长期滥用，在很多地区产生了耐药性虫株，严重制约了驱虫工作的有效开展。本项目拟在前期调查研究的基础上，对内蒙古地区绵羊主要消化道线虫丙硫咪唑耐药性产生情况进行调查；对丙硫咪唑耐药虫株和敏感虫株进行转录组、miRNA和lncRNA测序，运用生物信息学方法筛选可能的耐丙硫咪唑相关基因和miRNA、lncRNA，并对三者间的相互调控作用进行预测；再以qPCR、RNAi、构建线虫特异性表达载体和CRISPR/Cas9等技术对筛选的候选耐药基因、调控序列开展研究，探索线虫耐丙硫咪唑关键基因的功能和分子调控机制；并开展丙硫咪唑耐药性快速检测方法研究。研究成果可为绵羊寄生性线虫耐药机制、新药物靶点筛选和新兽药开发等相关研究奠定基础。

消化道线虫病是羊的主要寄生虫病，阿苯达唑（又称丙硫咪唑）等一些沿用多年的驱虫药在很多地区出现了耐药性，严重制约着驱虫工作的有效开展。本项目在内蒙古地区开展了绵羊消化道线虫及对阿苯达唑等驱虫药耐药性产生情况的调查；对优势虫种——捻转血矛线虫阿苯达唑耐药虫株和敏感虫株进行了全转录组测序分析，对阿苯达唑耐药相关基因、miRNA和lncRNA及三者间的相互调控作用等开展了相关研究。主要研究结果如下：. 调查证实内蒙古兴安盟科右前旗、乌兰察布察右后旗和鄂尔多斯乌审旗地区绵羊消化道线虫病仍很严重，三个地区绵羊消化道线虫平均感染率分别达到96.9%、63%和89%，优势虫种为捻转血矛线虫；对阿苯达唑均产生了明显耐药性。. 对捻转血矛线虫耐药虫株给药前后和敏感虫株样本进行了全转录组测序分析。给药前后虫株间筛选到851个显著差异DEGs；敏感虫株与耐药虫株间筛选到218个显著差异DEGs。对显著差异基因进行了功能注释和 KEGG富集分析。对ABC、GST和P450基因进行了原核表达和表达量检测。对捻转血矛线虫阿苯达唑耐药相关SNP多态性进行了分析。对HCON_00192020和I型β-tubulin等基因进行了多态性验证，并以5个耐药相关SNP基因建立了虫卵耐药性PCR检测方法。. 分别对捻转血矛线虫敏感虫株与耐药虫株、耐药虫株给药前与给药后进行了miRNA及miRNA-mRNA关联分析。在敏感虫株和耐药虫株间共发现294个差异显著的miRNA，预测到1770个miRNA-mRNA调控关系对。耐药虫株给药前后间共筛选到188个差异显著的miRNA，涉及353个miRNA-mRNA调控关系对。以双荧光素酶报告基因技术验证了hco-miR-5896与WDFY3、hco-miR-86与CAC 2个miRNA与其靶基因间的调控关系。. 捻转血矛线虫敏感虫株和耐药虫株间筛选到246个显著差异的lncRNA；对差异lncRNA与mRNA关联比较分析，获得91组靶向顺式调控关系，并对靶基因进行了功能注释和KEGG Pathway分析。. 分别对捻转血矛线虫阿苯达唑耐药相关基因GCY-12和GST基因进行了RNAi干扰和虫卵孵化抑制试验，实验证明了GCY-12和GST基因与阿苯达唑耐药性产生的相关性。.研究成果可为绵羊寄生性线虫耐药机制、新药物靶点筛选和新兽药开发等相关研究奠定基础。