Cloning Of The Rhomboid-Like Protease Genes In Eimeria.Tenella And Their Different Transcriptional Profiling During Development Stages

The Rhomboid-like proteases(ROMs) are members of a conserved family of intramembrane serine proteases in all eukaryotes and most prokaryotes. These intramembrane proteases are unusual polytopic enzymes and cleave their substrates proteins in or near transmembrane domains, which embed their water-dependent catalytic apparatus beneath the surface of the membrane. As known,the ROMs play roles in organsim diverse processes including growth fector signaling, quorum sensing in bacteria, mitochondrial membrane fusion, cell apoptosis, host cell invasion by pathogens and stem cell differentiation in eukaryotes. In apicomplexans, the known function of ROMs can process and maturate the organismal-origin proteins like micronems (MICs)、 rhoptry proteins(ROPs) and apicalmembrane antigens(AMAs) and take part in their host cell invasion.In this study, three genes that encode EtROMs were predicted from the genome date of Eimeria tenella using the bioinformatics tools, and amplified special primers using the designed base on three predicted coding sequences from sporozoite (Guangdong strain) total RNA template by RT-PCR. The sequencing results showed that the ORF lengths of EtROM1、EtROM3and EtROM4are864bp、774bp and1677bp, respectively. The bioinformatic analyses released that these ROMs include the complete rhomboid superfamily conserved domains and the seven typical transmembrane domains same as other species ROMs. Amino acid sequences alignment of EtROMs with other apicomplexan parasites by ClustalW showed these EtROMs shared21-60%idenficated in their amino acid comparisonThe parasites at different developmental stages were harvested and their total RNA were substracted by real-time PCR using Etactin gene as a reference for establishing standard curves to evaluate the copy number of three EtROMs genes. The results showed that all of quantative analyses were reliable because the melting curve showed a single peak and three genes have highest mRNA transcript levels in the sporozoite stage, and the lowest level during the unsporulated oosysts stage, so it is likely that these proteases can be translated and generously secreted in the process of invasion during the sporozoites stage. Two of them, EtROMland EtROM4, have similar expression levels and dynamic variations in all developmental stages of E.tenella. However, EtROM3has a different mRNA level from EtROM1and EtROM4, its transcriptional level of sporulated oosysts stage is much higher than2nd merozoites. This result states EtROM3is possible link to oosysts maturation except for invasion.