Comparative Transcriptome Analysis Of Three Development Stages Of Eimeria Necatrix And Cloning,Expression,and Functional Identification Of Differentially Expressed Genes

Coccidiosis caused by protozoan parasites of the genus Eimeria has a severe economic impact on commercial production worldwide.In China,annual costs for the control of coccidiosis are estimated to 30-60 million US dollars.Thus far,the conventional method to control the coccidiosis rely mainly on drugs,but there are many problems with this method,such as resistance and drug residues.Thus,more and more attention has been paid to developing new control methods.Eimeria necatrix has a high pathogenicity and mainly damages eight to eighteen-week-old chickens,and causes acute intestinal coccidiosis.The life cycle of Eimeria is consisted of three developmental stages including merogony,gametogony and sporogony.Merogony(asexual development)generally compose of three generations.It is generally believed that the development and sex differentiation of Eimeria depend on the differentially expressed genes and their regulation at different developmental stages.The isolation and purification of parasites at different stages are so difficult that restrict the development of coccidian molecular biology.There is few researches on the development of Eimeria at home and abroad.Especially,there is not a related report about the development of molecular biology of different developmental stages of Eimeria necatrix.In the life cycle of E.necatrix,the first and second generation of merogony occur in the epithelial cells of midintestine,while the third generation of merogony and gametogony occur in the epithelial cells of cecum.So the second-and third generation of merozoites can be easily separated and isolated.In the present study,the second-,third-generation of merozoites and gametocytes were isolated and purified,and their total RNA were extracted,respectively.Then the mRNA of the three developmental stages of parasites were sequenced using RNA-seq.Sequence datas were analyzed and annotated.Differentially expressed genes among the three developmental stages were identified and validated using real-time quantitative PCR(RT-PCR).GO and KEGG enrichment analysis were carried out for the putative biological function and pathways for the differentially expressed genes.Finally,four differentially expressed genes were expressed in vitro and identified using immunohistochemical technique.One recombinant protein was inoculated into chickens to evaluate its protective effect.In this paper,differentially expressed genes among three different development stages generated by RNA-seq and comparsion.The study of differentially expressed genes not only can decipher the molecular basis and mechanism of the growth and development of Eimeria necatrix,but also may find new drug and vaccine targets,so as to provide new ways and methods for the prevention and control of coccidiosis.1.Purification of the second-and third-generation merozoites and gametocytes of E.necatrixChickens were infected with the sporulated oocysts of E.necatrex by crop inoculation.And the second-generation merozoites were isolated from the mucosal tissues in the midintestine of the infected chickens.The average number of merozoites obtained from per chicken is 7.25×108 by the percoll density gradient centrifugation method.And then,the third-generation merozoites were isolated from the cecal mucosal tissue and purified using the DEAE-52 cellulose column.The average number of merozoites obtained from per chicken was 0.6×107.Additionally,the gametocytes were isolated from the cecal mucosa after 30 hours directly inoculating into the cecum with the second-generation merozoites.The gametocytes were purified by the percoll density gradient centrifugation,and 0.5×108 gametocytes were obtained from per chicken.2.Transcriptome analysis of the second-and third-generation merozoites and gametocytes of E.necatrixThe total RNA of purified parasites were isolated from the second-,third-generation merozoites and gametocytes respectively,and the sample libraries were constructed for the high throughput sequencing.The 68 009 332,51 854 332,73 219 844 raw reads were respectively obtained from the second,third-generation merozoites and gametocytes.After blasting,a total of 6 977 genes were annotated successfully in the second-generation merozoites,and 6 901 genes in the third-generation merozoites and 7 983 genes in gametocytes.Gene expression analysis showed that a total of 7 137 genes were expressed at three stages,71 genes were expressed only in the second-generation merozoites,and 61 genes for the third-generation merozoites and 340 genes for the gametocytes only.In the GO analysis,the number of GO terms was roughly same among three different development stages,however,the number of genes in the same GO term is different.KEGG analysis found that the pathways enriched among three development stages were different.The pathways of the second-generation merozoites took part in the splice,ribosome,synthesis,proteasome,DNA replication,RNA transport,etc.The enriched pathways for the third-generation merozoites were related to the Ribosome synthesis,RNA transport,RNA degradation,RNA polymeras and so on.The enriched pathways in the gametocytes were mainly synthesis of ribosomes,HIF-1 signaling pathway,splicosome,synaptosomal vesicle,synaptic vesicle circulation,lysosomal,pentose phosphate pathway and so on.3.Comparative transcriptome analysis between the second-and third-generation merozoites of E.necatrixThere were 2 053 differentially expressed genes between the second-and third-generation merozoites of E.necatrix,among which there were 1 261 up-regulated genes and 95 genes expressed only in the second-generation merozoites,while 837 up-regulated genes and 48 genes expressed only in the third-generation merozoites.GO enrichment analysis showed that 1 203 differentially expressed genes were successfully annotated by 59 GO terms,and 1091 differentially expressed genes were significantly enriched in 243 GO terms.In the KEGG pathway analysis,620 differentially expressed genes were successfully annotated by 198 signaling pathways,and 727 differentially expressed genes were significantly enriched in 242 signaling pathways.4.Comparative transcriptome analysis between the third-generation merozoites and gametocytes of E.necatrixThrough the comparative analysis of the expression level of each transcript between the third-generation merozoites and gametocytes,there were 4 267 genes significantly differentially expressed,among which 1 478 genes were significantly up-regulated expression and 329 genes were expressed only in the third-generation merozoites,while 2 789 genes were expressed up-regulated and 1 289 genes only in gametocytes.GO analysis of the differentially expressed genes showed that 1 295 genes were successfully annotated by 57 GO terms.KEGG pathway analysis indicated that 620 differentially expressed genes were successfully annotated by 198 signaling pathways.And the KEGG enrichment analysis showed that 725 differentially expressed genes were enriched in 241 signaling pathways.5.Prokaryotic expression of the differentially expressed genes and their immunofluorescence localizationAccording to the results of RT-PCR,four differentially expressed genes(EnPIPK,EnAGC,EnCK2 and EnHAP2)were selected,and their sequences were producted by synthesis or cloning after RT-PCR amplification,which were respectively inserted into the prokaryotic expression vector pET28a(+),and successfully expressed in E.coli BL21.The size of the fusion protein was 30 kDa,17 kDa,20 kDa and 20 kDa,and the four proteins mainly existed in a form of inclusion bodies.Western blot results showed that all four recombinant proteins could be specifically recognized by anti-6×HIS tag monoclonal antibody.Immunofluorescence results showed that EnPIPK,EnCK2 and EnHAP2 were located on the second-,third-generation meronts/merozoites and gametocytes,however,the fluorescence intensity of the proteins were different among three stages.EnAGC was only seen in the third-generation meronts/merozoites and gametocytes,not seen in the second-generation of meronts/merozoites.The results of fluorescence localization showed that the expressions of four genes at different developmental stages were consistent with those of fluorescence quantitative PCR.6.The immune protective effects of recombinant protein rEnHAP2After the recombinant protein rEnHAP2 purified and renatured,chickens were vaccinated with this recombinant protein at different doses and challenged with live parasites after the booster immunity except negative group.The immune protective effect of rEnHAP2 was evaluated on parasitological criteria(oocyst excretion,intestinal lesion scores and growth performance)and immunological response.Meanwhile,the expression level of the different cytokines induced was detected.There were two animal experiments.The results showed that rEnHAP2 had the best protective effect with 200?g immunization dose per chicken.The recombinant protein rEnHAP2 could decrease the oocyst yield and lesion score and improve the survival rate compared with non-vaccinated and infected control.Chickens immunized with rEnHAP2 also stimulated higher level of cells immune and humoral immunity.