The Primary Research Of Resistance Related Genes From Eimeria Tenella And Analysis Of Drug-resistant Strains And Sensitive Strain By Whole Genome Resequencing

Eimeria tenella(E.tenella)is a single cell parasitic protozoa parasitizing in chicken cecum and it is the major pathogen of chicken coccidiosis.At present,the prevention and treatment of chicken coccidiosis mainly rely on drugs.Long-term use of drugs has caused severe resistance to Eimeria spp and caused a lot of economic losses to the poultry industry.In order to study the mechanism of drug resistance of Eimeria,two drug-resistant strains which including diclazuril resistant strain(DZR)and maduramycin resistant strain(MRR)were induced according to E.tenella drug-sensitive strain(DS)in our lab.Transcriptome sequencing of two drug-resistant strains and drug-sensitive strain were performed to screen for differentially expressed genes.On this basis,four genes that were significantly up-regulated in two drug-resistant strains were selected in this study,including HD domain-containing protein(EtHDHD),glyceraldehyde 3-phosphate dehydrogenase(EtGAPDH),T-complex protein 1 subunit ?(EtTCP-1?)and malate dehydrogenase(EtMDH)of E.tenella.A preliminary analysis of the characteristics and functions of the four genes were performed.In order to verify the developmental mechanism of coccidia drug resistance,drug-sensitive strain and two drug-resistant strains were sequenced using whole genome resequencing.1.Cloning and bioinformatics analysis of EtHDCP,EtGAPDH,EtTCP-1? and EtMDH genesThe ORF sequences of EtHDCP,EtGAPDH,EtTCP-1? and EtMDH were successfully amplified by PCR using the first strand of sporulated oocyst cDNA of E.tenella.The length of EtHDCP ORF is 462 bp and encoding 153 amino acids.The molecular weight of the encoded protein is 16.8 kDa.The protein has no transmembrane structure and signal peptide.Functional domain structure analysis shows that it has mainly N-glycosylation site except for other domains.The length of EtGAPDH ORF is 1020 bp,encoding 339 amino acids.The molecular weight of the encoded protein is 37.3 kDa.The protein has no transmembrane structure and signal peptide.Functional domain structure analysis shows that it has mainly glyceraldehyde 3-phosphate dehydrogenase active site except for other domains.The length of EtTCP-1? ORF is 843 bp,encoding 280 amino acids.The molecular weight of the encoded protein is 30.8 kDa.There is no transmembrane structure and signal peptide.Functional domain analysis shows that the protein has mainly the chaperone molecule TCP-1 tag 2 site except for other domains and belongs to the TCP-1/cpn60 chaperone family.The length of EtMDH ORF is 954 bp.It encodes 317 amino acids and the molecular weight of the encoded protein is 33.9 kDa.There is no transmembrane region and signal peptide.Functional domain analysis shows that the protein has mainly lactate/malate dehydrogenase active site except for other domains.Quantitative PCR was used to detect the mRNA transcriptional levels of EtHDP,EtGAPDH,EtTCP-1? and EtMDH in four different developmental stages(Unsporulated oocysts(UO),Sporulated oocysts(SO),Sporozoites(SZ),Second-generation merozoites(SM))of E.tenella.The results showed that the transcriptional level of EtHDCP gene in SM stage is extremely high,and the transcriptional levels of other developmental stages are extremely low.The transcription of EtGAPDH can be detected in four developmental stages of E.tenella,and the transcriptional level from high to low is SM,UO,SZ,SO.The transcription of EtTCP-1? can be detected in the UO,SO and SM of E.tenella,and the transcriptional level from high to low is SM,UO and SO,but the transcript level is very low in the SZ stage.The transcription of EtMDH can be detected in four development stages of E.tenella,from high to low: SM,UO,SO,SZ.2.Expression of prokaryotic recombinant proteins and preliminary function analysis of EtHDCP,EtGAPDH,EtTCP-1? and EtMDHThe ORFs of EtHDCP,EtGAPDH,EtTCP-1? and EtMDH genes were ligated into the pGEX-4T-2,pCOLDI,pET-28 a and pGEX-4T-2 vectors respectively,and four recombinant plasmids,pGEX-4TEtHDCP,pCOLDI-EtGAPDH,pET-28a-EtTCP-1?,pGEX-4T-EtMDH were constructed.Four recombinant expression plasmids were identified by sequencing and then transformed into E.coli BL21 strain.The recombinant proteins GST-EtHDCP(rEtHDCP),HIS-EtGAPDH(rEtGAPDH),HIS-EtTCP?(rEtTCP-1?)and GST-EtMDH(rEtMDH)were successfully expressed by IPTG induction under different conditions.rEtHDCP,rEtGAPDH and rEtTCP-1? were expressed as inclusion body,while rEtMDH was expressed as soluble protein according to the analysis of the recombinant protein.The three inclusion body recombinant proteins were purified by cutting gel and the rEtMDH was obtained using GST purification column.New Zealand rabbits and BALB/c mice were immunized with purified rEtHDCP,rEtGAPDH,rEtTCP-1? and rEtMDH respectively to obtain the corresponding multiclonal-antisera.Western blot analysis showed that rEtHDCP,rEtGAPDH,rEtTCP-1? and rEtMDH had good reactionogenicity.Quantitative PCR and Western blot analysis showed that the expression of EtHDCP,EtGAPDH,EtTCP-1? and EtMDH were up-regulated in two drug-resistant strains.The enzyme activity analysis of EtGAPDH and EtMDH showed that the activity of GAPDH and MDH of two drug-resistant strains were higher than that of sensitive strain.Using indirect immunofluorescence mapped the distribution of EtHDCP,EtGAPDH,EtTCP-1? and EtMDH at different developmental stages of E.tenella.The results showed that EtHDCP mainly located on the surface of SZ and SM stages.After SZ added to DF-1 cells for 48 h,protein expression of EtHDCP was increased;EtGAPDH mainly localized on the surface of the SZ and the cytoplasm of the SM;SZ added to DF-1 cells after 48 h,the expression level was increased;EtTCP-1? was mainly located on the surface of the parasite.After SZ was added to DF-1 cells for 24 h,it was distributed in the whole body except for the refractive body.EtMDH was widely distributed in the SZ except for the refractive body and the surface of SM stage.When the SZ was added to DF-1 cells for 2 h,the EtMDH protein was mainly located at the top of the sporozoites.After SZ was added to DF-1 cells for 48 h,the protein expression increased.Through inhibition experiments in vitro,it was found that the sporozoite invasion ability was significantly decreased after incubated with the antirEtHDCP and anti-rEtMDH antibodies compared with the control group.When the antibody concentration rised to 400 ?g/mL,the inhibition rate of rEtHDCP antibody exceeds 40%,and the inhibition rate of rEtHDCP antibody is about 40%.3.SNP Screening for sensitive and resistant strains based on whole genome resequencingSporulated oocysts of DS,DZR and MRR of E.tenella were collected and purified.The DNA of three strains of E.tenella were extracted and analyzed by whole genome resequencing.The clean data were obtained after filtering raw data.Using the Fst and sliding window methods in PoPoolation 2 software,we obtained 45 drug-related genes at a coverage depth of 40×.GO analysis and KEGG analysis showed that these drug-related genes are involved in the biological processes,cell composition and molecular function.Further comparative analysis found that the mutations of the following genes were absent in the sensitive strains,but existed in resistant strains: acetyl-CoA carboxylase(ACC)50424C>S,50438G>K,50443G>R,51264A>R,51321C>S,calmodulin-dependent 3',5'-cyclic nucleotide phosphodiesterase(CaM-PDE)619583G>K,619603C>S,619605A>R,619607G>S.In addition,mutations in the following gene is not present in resistant strains,but in sensitive strain: heat repeat family protein(HRFP)2164C>Y,2170T>W and 2173G>R.