Analysis And Identification Of Related Resistant Genes Of Eimeria Tenella

The universality and severity of the development of drug resistance of Eimeria has become a major obstacle in the prevention and treatment of coccidiosis.Reserchers have studied the causes and mechanisms of Eimeria resistance to different anti-coccidia drugs.The mechanism of the development drug resistance is unknown yet,the target genes also have not been identified,so the molecular mechanism of drug resistance toEimeria was unclear.In order to further study the mechanism of drug resistance of Eimeria,two drug-resistant strains of E.tenella,maduramicinresistant(MRR)strain and diclazuril-resistant(DZR)strain were induced,the comparative transcriptome analyses of a drug-sensitive strain(DS)and two drug-resistant strains(MRR and DZR)of E.tenella were performed by RNA-seqencing in our previous study.Some differentially expressed genes(DEGs)were identified in the comparison of two drug-resistant strains vs drug sensitive strain.Based on this study,three DEGs were selected and identified,including the up-regulated gene NADP-specific glutamate dehydrogenase(NADP-GDH)and superoxide dismutase(SOD)in two drug-resistant strains,one the down-regulated gene surface antigen 10(SAG10).1.Cloning and bioinformatics analysis of three genesThree genes of E.tenella,surface antigen 10(EtSAG10),NADP-specific glutamate dehydrogenase(EtNADP-GDH)and superoxide dismutase(Et SOD),were cloned using the first strand of E.tenella sensitive strain cDNA successfully and analyzed by bioinformatics technology.The results showed that the length of EtSAG10 open reading frame sequence was 786 bp,it encoded a protein of 261 amino acid residues with a predicted molecular mass of 27.9 kDa and an isoelectric point of 4.7.Signal peptide and transmembrane structure analysis indicated that the protein had a signal peptide and a transmembrane structure and contained multiple functional sites.EtSAG10 belonged to the surface antigen family(SAGs).The length of EtNADP-GDH open reading frame sequence was 1407 bp,it encoded a protein of 468 amino acids residues with a predicted molecular mass of 51.0 kDa and an isoelectric point of 6.93.Signal peptide and transmembrane structure analysis indicated that the protein encoded by this gene had no signal peptide and transmembrane structure,and contained a variety of functional sites including NAD binding sites.The length of EtSOD open reading frame sequence is 651 bp,it encoded a protein of 216 amino acids residues with a predicted molecular mass of 24.9 kDa and an isoelectric point of 7.15.Signal peptide and transmembrane structure analysis indicated that the protein encoded by this gene has no signal peptide and no transmembrane structure,but included multiple functional sites.2.Prokaryotic expression and characteristics of three genesThe three genes were cloned into the prokaryotic expression vector,respectively.The three recombinant expression plasmids,pGEX-4T-EtSAG10,pColdI-Et NADP-GDH and pET-28a-EtSOD,were constructed and transformed into E.coli BL21 competent cells,the recombinant proteins,GST-Et SAG10(rEt SAG10),His-EtNADP-GDH(rEtNADP-GDH)and His-EtSOD(rEtSOD),were induced to expression successfully and obtained,and all three recombinant proteins were soluble proteins.New Zealand white rabbits and BALB/c mice were immunized with recombinant protein to prepare corresponding polyclonal antibodies.Immunofluorescence localization and invasion inhibition experiments analyzed the distribution of three proteins in the parasites and whether they were involved in the invasion of host cells by sporozoite.The localization analysis found that EtSAG10 was mainly distributed on the surface of the parasites and the parasitophorous vacuole membrane(PVM).With the development of parasites in DF-1 cells,the expression level of EtSAG10 increased.EtNADP-GDH is mainly distributed on the surface and cytoplasm of parasites.With the development of parasites in DF-1 cells,the fluorescence was decreased and the expression of EtNADP-GDH also was decreased,but the expression of mature schizonts increased significantly.EtSOD is mainly distributed on the apical and surface of the parasites after invading host cells.With the development of intracellular parasites,EtSOD is located on the entire surface of immature schizonts and mature schizonts.Invation inhibition assay showed that both EtSAG10 and EtNADP-GDH were associated with sporozoite invasion of host cells,and the inhibition rate reached 60% and 45%,respectively,while EtSOD was nearly not associated with invasion.Transcriptional analysis of four developmental stages of E.tenella showed that EtSAG10 had the highest level of transcription in the sporulated oocyst stage,EtNADP-GDH had the highest level of transcription in the unsporulated oocyst stage,in contrast,EtSOD had the highest level of transcription in the second generation merozoite stage.3.Differences of three genes between sensitive and resistant strains of E.tenellaThree strains of E.tenella including sensitive strain,diclazuril-resistant strain and maduramycin-resistant strain,were collected,and the transcription levels and translation levels of the three genes were analyzed.The results showed that the transcriptional level and translational level of EtSAG10 in the two resistant strains were significantly down-regulated.The detection of E.tenella resistant strains induced by different concentrations of diclazuril showed that the translation level showed irregular changes,and the expression increased firstly and then decreased with the increase of drug concentration.Moreover,under the different concentrations of maduramycin,the translation level also increased firstly and then decreased.The transcriptional level and translation level of EtNADP-GDH in the two resistant strains and susceptible strains were not significantly changed in general,and did not cause significant changes in the transcription level or translation level of the gene under the action of different concentrations of drugs.The transcription level and translation level of EtSOD in two drug-resistant strains were significantly up-regulated.Further studies found that the detection of E.tenella resistant strains induced by different concentrations of diclazuril showed that the transcription level and translation level of the gene were positively correlated with drug concentration.The same results were also found in the analysis of E.tenella resistant strains induced by using different concentrations of maduramycin.In addition,enzyme activity analysis of EtNADP-GDH and EtSOD showed that the activity of EtNADP-GDH did not change significantly in the three strains,but the enzyme activity of Et SOD increased in the two resistant strains significantly.