Molecular Characterization Of Serine/threonine Protein Phosphatase And ATPase ASNA1 Homolog Of Eimeria Tenella

Coccidiosis is a serious intestinal parasitic disease caused by several types of Eimeria spp.At present,the widespread use of anticoccidial drugs has exacerbated the resistance of Eimeria spp to drugs.In order to further study the molecular mechanism of drug resistance,we have obtained diclazuril-resistant(DZR)strains and maduramicin-resistant(MRR)strains by inducing E.tenella drug-sensitive(DS)strain from low to high concentraion in our lab and carried out the comparative transcriptome analyses of DS strain and two drug-resistant(DZR and MRR)strains of E.tenella by RNA-seqencing in our previous study.In this study,two genes that were significantly up-regulated in both drug-resistant strains compared to the DS strains were analyzed.They were E.tenella serine/threonine protein phosphatase(EtSTP)and ATPase ASNA1 homolog of E.tenella(EtASNAl).1.Cloning and expression of EtSTP and EtASNAlUsing the first strand of the cDNA of E.tenella DS strains as a template,EtSTP and EtASNAl was successfully cloned by PCR.Nucleotide sequence analysis showed that the 528-bp open reading frame of EtSTP encodes a polypeptide of 175 amino acid residues,and the predicted molecular mass is 19.0 kDa.The 408-bp open reading frame sequence of EtASNAl encodes a polypeptide of 135 amino acid residues,and the predicted molecular mass is 14.6 kDa.We connected the ORF to the prokaryotic expression vector pGEX-6p-1,and successfully constructed the prokaryotic recombinant expression plasmids to transform E.coli BL21.After that,the recombinant proteins rEtSTP and rEtASNAl were obtained by IPTG.Polyclonal antibodies against recombinant proteins were successfully obtained by immunizing New Zealand white rabbits and B ALA/c mice with the purified recombinant proteins.2.Functional analysis of EtSTP and EtASNAl in E.tenellaWe analyzed the expression levels of EtSTP and EtASNAl at different developmental stages of E.tenella using qPCR and Western Blot.The results showed that transcription levels and translation levels of EtSTP and EtASNAl were higher in unsporulated oocysts(UO)and second-generation merozoites(SM)than in sporulated oocysts(SO)and sporozoites(SZ).Indirect immunofluorescence localization was used to analyze the distribution of EtSTP and EtASNAl in parasites.The results showed that EtSTP was located in most areas of the SZ with the exception of refractile bodies.After the invasion of DF-1 cells by SZ,fluorescence intensity was enhanced,expression levels increased,and the protein localized to the parasitophorous vacuole membrane(PVM).The localization of EtASNAl showed that the protein was always present in most areas of the parasites.In vitro inhibition experiments showed that the ability of SZ to invade cells was significantly decreased after treatment with anti-rEtSTP antibody,and the inhibition rate could reach 32%,while anti-rEtASNAl also inhibited 20%of SZ from invading DF-1 cells.3.Differences between EtSTP and EtASNAl genes in sensitive and resistant strains of E.tenellaTransmission electron microscopy was used to observe the ultrastructure of SM of sensitive and resistant strains,and it was found that the starch granules in the resistant strains increased significantly.Three strains of E.tenella,including DS,DZR and MRR strains,were collected,and the transcription levels and translation levels of the two genes were analyzed.The results showed that the transcriptional level and translational level of EtSTP and EtASNAl in the two resistant strains were significantly up-regulated.qPCR was used to analyze the resistant strains of diclazuril E.tenella isolated from the field.The transcription levels of wild resistant strains isolated from the field were also significantly higher than DS strains.The qPCR was used to detect the mRNA transcription levels of DZR and MRR strains at different concentrations.It was found that the transcription level increased gradually with the increase of drug concentration.In addition,in vivo and in vitro tests were used to analyze the expression levels of EtSTP and EtASNAl under the action of the drugs,and it was found that the addition of the drugs would cause the expression to be up-regulated.This study provides a basis for further research on the molecular mechanism of drug resistance and identification of drug-resistant molecular markers.