| Coccidiosis is a serious parasitic disease in the intestinal tract of parasitic chickens.At present,the disease mainly depends on the prevention and treatment of drugs.However,the long-term and widespread use of drugs led to serious drug resistance of coccidian.In order to study the molecular mechanism of drug resistance of Eimeria tenella,the whole genome of drug sensitive strains and salinomycin resistant strains were sequenced.Selective sweep was used to screen candidate genes that may be related to salinomycin resistance of E.tenella.The characteristics of three drug resistant related genes(E.tenella conserved protein 35(EtCHP35),E.tenella conserved protein 40(EtCHP40)and E.tenella SAG family member(EtSAG))were analyzed.1.Screening of salinomycin resistant-related genes by Whole genome resequencing and Selective sweepThe whole genome of 10 E.tenella drug sensitive strains,3 laboratory induced E.tenella salinomycin resistant strains with different concentrations,and 15 E.tenella salinomycin resistant strains isolated from the field by using a single oocyst method were sequenced.The results showed that the sample data were all above 1Gb,Q20 ≥ 95%,Q30 ≥ 90%.The comparison results showmore than82% of clean data were papped onto the reference genome,the sequencing Average depth is more than14%,and the coverage(4 ×)is higher than 92%.The datas of resequencing of drug-sensitive and salinomycin-resistant strains of E.tenella were analysized by selective sweep method,1221 SNPs were obtained from 73 selected genes in sensitive strains,of which 81 SNPs were non-synonymous mutations.And 2545 SNPs were selected in salinomycin resistant strains,of which 118 SNPs were non-synonymous mutations.GO enrichment analysis showed that the candidate genes of sensitive strains were mainly involved in biological processes and molecular functions,while those of salinomycin resistant strains were mainly involved in biological metabolism.KEGG pathway analysis showed that the candidate genes of sensitive strains were mainly involved in phagosome,oxidative phosphorylation and metabolism,while those of salinomycin resistant strains were mainly involved in phagosome and metabolism.14 genes were obtained by correlation analysis with the transcriptome results and the candidate genes for selective sweep,which were mainly involved in ribosome and ATP transport pathways.2.Cloning and bioinformatics analysis of three drug resistant related genesUsing the first strand of c DNA of sporulated oocysts of E.tenella as the amplification template,the ORF sequences of EtCHP35,EtCHP40,and EtSAG were cloned successfully.Bioinformatics analysis showed that the protein encoded by the three genes has transmembrane structure and has multiple protease phosphorylation sites.EtCHP35 contains a reverse transcriptase site and EtCHP40 contains an aspartic protease active site.EtSAG contains a signal peptide and a threonine-rich region,which belongs to the SAG family member.The prokaryotic recombinant expression plasmids were successfully constructed and three recombinant proteins(r EtCHP35,r EtCHP40 and r EtSAG)were obtained,and the corresponding antibodies were prepared,respectively.3.Analysis of the characteristics of three drug resistant-related genesThe results of indirect immunofluorescence localization showed that EtCHP35 and EtCHP40 were mainly distributed on the surface and anterior of sporozoite,while EtSAG was mainly distributed on the surface of sporozoite and the second generation merozoite.In vitro inhibition experiments showed that these three proteins were not involved into the invasion of sporozoites into host cells.Real-time fluorescence quantitative PCR was used to analyze the mRNA transcription level of E.tenella sensitive strain at different developmental stages and different strains(drug-resistant and sensitive strains).The results showed that the mRNA transcription levels of EtCHP35 and EtSAG genes were highest in the sporulated oocysts of the sensitive strain.EtCHP40 showed that the expressed level was higher in the second generation merozoites than other three stages.Compared with the sensitive strains,the mRNA transcription level of EtCHP35 gene was up-regulated in the second generation merozoites of maduramycin resistant strains and salinomycin resistant strains.The EtCHP40 was up-regulated in the second generation merozoites of the three drug-resistant strains,and the transcriptional level of maduramycin-resistant strains was significantly higher than that of other drug-resistant strains.The mRNA transcription level of EtSAG was up-regulated in maduramycin-resistant strains and down-regulated in salinomycin-resistant and diclazuril-resistant strains in the sporulation oocysts.Moreover,the mRNA transcription levels of EtCHP35 and EtCHP40 were increased with the increase of salinomycin concentration.These results provide a basis for further studying the molecular mechanism of E.tenella drug resistance and establishing a rapid detection method for coccidia drug resistance in the field. |
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