Characterization And Annotation Of Babesia Orientalis Apicoplast Genome And The Function Of RON2 In Invasion

Babesia orientalis, as a protozoon species infecting water buffalo, is transmitted by Rhipicephalus haemaphysaloides. It is prevalent in many provinces to the south of Yangtse River in China. The clinic manifestations are fever, anemia, icterus, hemoglobinuria and frequent mortality, and this pathogen causes enormous economic losses in breeding industry. Until now, the knowledge of Babesia orientalis genome information is quite limited; the invasion mechanism is also fewly reported.In the present study, the full length of B. orientalis Apicoplast genome sequence was cloned, sequenced and annotated. The overall characteristic of the structure was carried on the detailed comparative analysis. Another hand, we obtained the information of Rhoptry neck protein 2,and studied the interaction between RON2 and Apical membrane antigen 1 preliminary.(1) Sequencing and Analyzing the Apicoplast genome of B. orientalisFor cloning Apicoplast genome of B. orientalis, according to high-throughput sequencing and cDNA library of B. orientalis genome,we obtained partial contigs belonging to Apicoplast genome, specific primers were designed from the conserved region of Apicoplast sequences of hemoparasites in NCBI,cloned and sequenced the full length Apicoplast genome of B. orientalis. Assembled and annotated with different software and Artemis. The complete genome map indicated that it encoded a circular DNA(78.9%A+T) of 33.2 kb in size. The apicoplast genome unidirectionally encodes a large- and a small-subunit ribosomal RNAs, 24 species of different tRNA, four DNA-dependent RNA polymerase beta subunits(rpoB, rpoC1, rpoC2 a and rpoC2b), 17 ribosomal proteins, and a EF-Tu elongation factor, 2 Clp protease chaperone genes, 14 hypothetical proteins. Furthermore, genome analysis and annotation revealed a similar gene structure and organization of the B. orientalis apicoplast genome compared with B. bovis.Further, the detailed comparative analysis was performed between B. orientalis apicoplast genome and related parasites in NCBI. Comprehensive results showed that the apicoplast genome of B. orientalis is more closed to B. bovis in the genome size, the gene species, the gene organizations, the order of genes and gene phylogenetic analysis.(2) The interaction between RON2 and AMA1 proteinAccording to the RON2 sequences reported on the NCBI, specific primers were designed to clone RON2 gene of B. orientalis. A 4035 bp full length RON2 gene from B. orientalis genome was amplified. In this study,prokaryotic expression plasmids of the short length RON2(p GEX-KG-RON2-2, pGEX-KG-RON2-5)interact with AMA1 were successful constructed and expressed in E. coli. Far-western and the adhesion ELISA were performed with purified RON2-2, RON2-5. The results showed that the interaction between the recombinant protein of RON2-2/RON2-5 and natural AMA1 is existence in certain proportion.