柔嫩艾美耳球虫抗马杜拉霉素、地克珠利虫株特异消减文库.PDFVIP

中国农业科学 2005,38(8):1712-1716 Scientia Agricultura Sinica 柔嫩艾美耳球虫抗马杜拉霉素、地克珠利虫株 特异消减文库的构建 韩红玉, 赵其平, 姜连连, 董 辉, 陈兆国, 黄 兵 （中国农业科学院上海家畜寄生虫病研究所/农业部动物寄生虫学重点开放实验室，上海 200232 ） 摘要：以遗传背景一致的柔嫩艾美耳球虫（Eimeria tenella）敏感株孢子化卵囊为驱动组，马杜拉霉素抗 药株和地克珠利抗药株孢子化卵囊为试验组，利用抑制消减杂交（SSH）方法，通过两轮杂交和两次PCR 分别构建 了富含两个抗药株孢子化卵囊与敏感株孢子化卵囊之间差异表达基因的消减cDNA 文库。随机从两个消减文库中分 别挑取 50 个克隆，经 PCR 鉴定马杜拉霉素抗药株和地克珠利抗药株孢子化卵囊的消减文库重组率分别为 96%和 98%，差异基因片段大小分布在250 bp～1.0 kb 之间。从每个文库中随机挑取65 个阳性克隆经斑点杂交试验，分 别选择表达量上调的6 个克隆进行序列分析和同源性比较。结果发现，有4 个cDNA 片段可能是新基因片段，与地 克珠利抗药株的产生有关；有 3 个新的 cDNA 片段可能与马杜拉霉素抗药株的产生有关。这些结果将为克隆全长 cDNA 和探索球虫抗药性产生及发展的分子机理奠定了一定的基础。 关键词：柔嫩艾美耳球虫；马杜拉霉素；地克珠利；抗药性相关基因；抑制消减杂交；基因克隆 Construction of Subtractive cDNA Libraries of Eimeria tenella to Maduramycin and Diclazuril by Suppression Subtractive Hybridization HAN Hong-yu ，ZHAO Qi-ping ，JIANG Lian-lian ，DONG Hui ，CHEN Zhao-guo，HUANG Bing (Key Laboratory for Animal Parasitology, Ministry of Agricultry/Shanghai Institute of Animal Parasitology， Chinese Academy of Agricultural Sciences, Shanghai 200232) Abstract: In order to clone and identify differentially expressed genes in Eimeria tenella relating to drug resistance to Diclazuril and Maduramycin, the cDNA from sporulated oocysts of the drug-sensitive strain of E.tenella was used as driver and the cDNAs from sporulated oocysts of two drug-resistant strains were used as tester to construct subtractive cDNA libraries using the technique of suppression subtractive hybridization(SSH). PCR amplification revealed that the two subtractive cDNA libraries of Maduramycin and Diclazuril-resistant strains of E.tenella contained approximated 96% and 98% recombinant clones, respectively. Plasmid ins