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

 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。

Construction of Subtractive cDNA Libraries of Eimeria tenella to Maduramycin and Diclazuril by Suppression Subtractive Hybridization

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 inserts were PCR amplified and the lengths were 250 bp-1.0 kb. The results of dot-blot hybridization showed that six clones were over-expressed in the drug-resistant strains. Sequence analyses revealed that three and four cDNA fragments were likely to represent novel genes relating to resistance to Maduramycin and Diclazuril, respectively. These results have provided the foundation for cloning full-length genes and studying molecular mechanism relating to drug-resistance in E.tenella.