【摘要】 以柔嫩艾美耳球虫(E im eria tenella)敏感株、地克珠利抗药株和马杜拉霉素抗药株孢子化卵囊为材料,用银染mRNA差异显示方法筛选和克隆与球虫抗药性相关的基因。首先提取这3个虫株孢子化卵囊的总RNA为模板,用O ligo(dT)12GG为锚定引物和2个10碱基随机引物组合进行RT-PCR,产物经变性聚丙烯酰胺凝胶电泳后银染。分别切取5条差异条带,进行2次PCR扩增,产物回收后与PGEM-T-easy V ector连接转化。经PCR鉴定正确后,再进行斑点杂交试验、序列分析和同源性比较。结果发现,地克珠利抗药株和马杜拉霉素抗药株分离的差异片段中都有2个cDNA片段(可能为新的基因片段),这为克隆全长cDNA和探索球虫抗药性产生的分子机理奠定了一定的基础。更多还原

【Abstract】 Silver-staining mRNA differential display method was used to isolate and clone the differential expressed gene of drug-resistant strains of Eimeria tenella to diclazuril and maduramycin.Total RNA of three strains of E.tenella was isolated by using Trizol.DDRT-PCR was established by using an anchored primer Oligo(dT) 12GG and two arbitrary primers.Their products of PCR were analyzed on the denaturing polyacrylamide gels by silverstaining.The differential cDNAs fragment was excised from the gels and reamplified with the same sets of primers.Then the PCR products were purified and ligated with PGEM-T-easy Vector.The dot-blot hybridization proved that the two clones from each of two drug-resistant strains of E.tenella appeared the strong signals.Sequence analysis revealed that the two cDNA fragments would be a novel genes.This study paved the way for cloning the full-length cDNAs and finding the molecular mechanism about the drug-resistance of E.tenella.更多还原