| Coccidiosis caused by protozoan parasites of the genus Eimeria has a severe economic impact on commercial poultry production worldwide. Coccidiosis is distributed widely and occurs throughout the year. Currently, coccidiosis is mainly prevented by adding anticoccidial drugs in the feed in every country, applying complementally with therapeutic drugs and live coccidia vaccine. Currently, drug resistant coccidian strains distribute all over the world, most of them are multi drug resistance and cross resistance. Drug resistant coccidia reduces economic benefits in poultry industry dramatically. So far, the mechanism of drug resistance is still in the theory stage, and there are not drug resistance genes has been reported.With the cloning and expressing technology of parasite-specific antigen gene, people research genes which about drug resistance of the coccidia by constructing cDNA library at the molecular level, in order to understand the mechanism of drug resistance further. The suppression subtractive hybridization technique is one of the effective methods to clone differentially expressed genes in cDNA rapidly. We can identify genes which are bound with the growth and development of the parasite and other differentially expressed genes which are related to the drug resistance using SSH technology, and study the mechanism of drug resistance. EST is a part of cDNA fragments of these expressed genes. Compared with the whole genome sequences, EST sequences have some advantages which are rapid, cheap and acquirement easily.Our lab has sequenced and initial bioinformatics analyzed subtractive libraries of two drug resistance strains of E. tenella on the basis of subtractive cDNA libraries which constructed by suppression subtractive hybridization technique and have conformable genetic background and contain different express genes between the Diclazuril resistant strains and Maduramycin resistant strains sporulated oocysts and sensitive strains sporulated oocysts of E. tenella.We successfully obtained 568 EST from subtractive libraries of Diclazuril resistant strains. After splicing and clustering, we obtained 107 Unigenes which contain 44 singlets, 63 contigs, 12.1% of the Unigenes are high redundancy rate express genes which EST appearance 8 times. The average length of EST is 605 bp, 97.2% of the EST are longer than 300 bp which 58.9% are 500～900 bp. 13 Unigenes have poly (A) construction and 5 Unigenes have poly (T) construction. After homology aligned with databases, we found that 82.2% (0≤E≤e-100) genes are highly homology genes, 10.3% (E>e-20) are unknown genes, and 3 genes haven't homology match genes. We obtained some functional annotations of 72 Unigenes after scoured UniProt database which origined from 11 species, 39.3% Unigenes origin from Toxoplasma gondii, but only 11.2% origin from E. tenella. Besides, 66 Unigenes have GO functional classification which belong to living being process (17), celelular component (7) and molecular function (42). After analyzed by ESTScan, we found 89 ORF which haven't stop codon and longer than 300bp. After scour COGs, 23 Unigenes are classified among proper COG family.We successfully obtained 475 EST from subtractive libraries of Maduramycin resistant strains. After splicing and clustering, we obtained 87 Unigenes which contain 45 singlets, 42 contigs, 19.5% of the Unigenes are high redundancy rate express genes which EST appearance 8 times. The average length of EST is 615 bp, 90.8% of the EST are longer than 300 bp which 51.7% are 500～900 bp. 11 Unigenes have poly (A) construction and 2 Unigenes have poly (T) construction. After homology aligned with databases, we found that 70.1% (0≤E≤e-100) genes are highly homology genes, 19.6% (E>e-20) are unknown genes , and 7 genes haven't homology match genes. We obtained some functional annotations of 63 Unigenes after scoured UniProt database which origined from 11 species, 60.3% Unigenes origin from Toxoplasma gondii, but only 17.5% origin from E. tenella. Besides, 58 Unigenes have GO functional classification which belong to living being process (11), celelular component (6) and molecular function (41). After analyzed by ESTScan, we found 68 ORF which haven't stop codon and longer than 300 bp. After scour COGs, 19 Unigenes are classified among proper COG family.In summary, this study find a crop of novel genes after sequenced and analyzed 2 subtractive libraries which constructed with coccidian drug resistance strains and sensitive strains which have conformable genetic background. The annotation and bioinformatics analysis about these genes maybe provide research thought for researching mechanism of drug resistance of coccidian, constructing molecular detection of the drug resistance to the 2 medicine of E. tenella, preventing and delaying production of the drug resistance. This study also develops genomics research of coccidian, and provides groundwork for preventing and controlling coccidiosis.
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