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250bp～1.0kb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。
In order to clone and identify differentially expressed genes in Eimeria tenella relating to drug resistance to Dielazuril and Maduramyein, the eDNA from sporulated ooeysts of the drug-sensitive strain of E.tenella was used as driver and the eDNAs from sporulated ooeysts of two drug-resistant strains were used as tester to construct subtraetive eDNA libraries using the technique of suppression subtraetive hybridization（SSH）. PCR amplification revealed that the two subtraetive eDNA libraries of Maduramyein and Dielazuril-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 Maduramyein and Dielazuril, respectively. These results have provided the foundation for cloning full-length genes and studying molecular mechanism relating to drug-resistance in E.tenella.
出处
《中国农业科学》
CAS
CSCD
北大核心
2005年第8期1712-1716,共5页
Scientia Agricultura Sinica
基金
国家自然科学基金资助项目(30000124)
国家科技攻关资助项目(2002BA514A-17-06)
