Structural And Functional Characterization Of Proteasome β5Subunit Encoding Gene Of Haemonchus Contortus

Haemonchosis of ruminant has been an important factor limiting livestock animal production. Haemonchus contortus is an important ruminant gastrointestinal parasitic nematode with a worldwide distribution. H. contortus mainly are living in abomasum and small intestine of sheep and cattle, causing anemia and malnutrition, sometimes even resulting in host death. Due to the lack of available vaccines, anthelmintic is the primary means of treatment. However, the uncontrolled use of medicine has resulted in the world-wide resistance. Hence, exploring new ways to control the disease is urgent. Understanding the growth and reproduction adjustment mechanism of H. contortus at the molecular level can help the development of new drugs and discover potential candidate vaccine’s candidate target molecules.Proteasome is a highly conserved multi-subunit enzyme complex, widely present in eukaryotic cells. Proteasome participate in a variety of cell functions, including cell cycle and apoptosis regulation, increased antigen presentation, etc. Proteasome is also involved in the growth and regulation of parasites, and induces host immune response. Research on the proteasome is also continuously made new achievements successively found the presence of proteasome in Plasmodium, Leishmania, Amoeba, Toxoplasma gondii, Schistosome and other parasites, and play an important role in the biology of these parasites. However, almost nothing is known about the nematode proteasomes.Lack of effective means of culture in vitro hampers the establishment of technology platforms for studying gene function. C. elegans as a classic model organism has a complete genomic information and free life cycle. It is easy to be maintained in the laboratory and has very close genetic relationship with H. contortus.In the present study, the H. contortus proteasome05subunit encoding gene (Hc-pbs-5) was isolated and characterized by bioinformatics analysis. The transcription levels of Hc-pbs-5in various developmental stages of H. contortus was analyzed by RNAseq analysis and RT-PCR. heterologous RNAi method was used to analyze the function of Hc-pbs-5gene, evaluating its functional conservation with Ce-pbs-5gene. Use heterologous transgenic method was also attemped to analyze the expression patterns of Hc-pbs-5promoter in C. elegans. Results as follows:1. Isolation of Hc-pbs-5full-length cDNA sequenceOn the basis of the ESTs of Hc-pbs-5gene, we obtain the Hc-pbs-5cDNA sequence by RACE method. Bioinformatics analysis revealed:Hc-pbs-5cDNA sequence is1117bp in length, encoding284amino acids. The Hc-PBS-5protein is highly homologous with PBS-5family. H-pbs-5has a typical PTS_HPr domain. Full-length gDNA2310bp in size, containing7exons and6introns, all exons and introns follow the GT-AG rule.2. The transcriptional profiles of Hc-pbs-5in different developmental stages of H. contortusThe transcription of Hc-pbs-5was analyzed by RNAseq and RT-PCR analysis in different developmental stages of H. contotrtus including eggs, the first-stage larvae, the second-stage larvae, the third-stage larvae, the fourth-stage females, the fourth-stage males, female and male adult worms. The transcription was at the highest level in eggs, with gradual decreasing in the free-living stages with the lowest level at L3larvae, and then recovery increase in parasitic stages.3. Hc-pbs-5gene and Ce-pbs-5gene has functional similarityUse E. coli HT115as a vector, the dsRNA was produced in C. elegans, use Ce-pbs-5gene as a positive control to assess the rate of oviposition and egg incubation of interferred strain respectively as well as the rate of diapause within24h of interference. The results showed that the rate of larval diapause by Hc-pbs-5dsRNA interference increased significantly, which is consistent with that of the positive control.4. Analysis on the promoter region of Hc-pbs-5gene and its localization in C. elegans.The upstream and5’-UTR sequences of Hc-pbs-5gene was discovered by database search. Bioinformatics analysis revealed that Hc-pbs-5is a second gene in the operon. Therefore its promoter is the promoter of the upstream gene. To study the gene expression patterns, two plasmids with fusion exprotein GFP was built. One comprises the promoter, and another has the segment between Hc-pbs-5and upstream gene. The two plasmids were injected into wild-type C. elegans, but they failed to show GFP localization.In summary, this study obtained H. contortus Hc-pbs-5full-length cDNA and gDNA sequence, and predicted the promoter DNA sequences. It is found that the gene is highly conserved with homologues from other organisms. The gene has the highest transcription in eggs, suggesting that it may be related to the growth and development of H. contortus. The results of heterologous RNAi also support this conclusion. The result from the study laid a solid foundation to further study on the functional role of Hc-pbs-5in regulating the development of parasitic nematodes.