| The gastrointestinal nematode Haemonchus contortus is a major blood feeding pathogen of sheep and goats, causing a great economic loss to the agricultural industry worldwide. Diapause is strategy to adapt this nematode to hostile environmental conditions and the key reason for "spring rise". Therefore, the research on diapause is essential for preventing and controlling the disease. Diapausing larvae of H. contortus ZJ strain were obtained and characterized from abomasums of sheep according to Gibbz (1971). The complete cDNA and gene of Hc-fau were isolated through RACE and Genome walking. Analysis of the regulating role on development was conducted by microinjection and RNAi in C. elegans. Research on functions and relationgship between domains of Hc-FAU was performed through transfection in HEK293 cell line. The Hc-FAU protein structure was modeled through homology modeling with SwissModel. These results will pave for reseaching on the diapause mechnasm.1. Characterizion of diapause larvae of H. contortus ZJ strainThe larvae were characterized by similar length, measuring 1067±97 μm (n=18) and rod-like shape of crystalline inclusion in the intestinal cells. Whole animal DAPI staining revealed genital primordium and the crystalline inclusion in diapause, indicating the crystalline inclusion might contain nucleic acid. The low temperature might not be the key initiated factor inducing diapause through temperature analysis.2. Obteining the Hc-fau and its expression patternIn this study, we identified a new gene Hc-fau, a homologue of human fau and C. elegans Ce-rps30. Hc-fau encodes two proteins through alternative RNA splicing, Hc-FAUA and Hc-FAUB consisting of 130 and 107 amino acids, respectively. Hc-FAU possesses a diverged ubiquitin-like (UBiL) protein domain and a conserved ribosome protein S30 domain. The protein is ubiquitously expressed, except in the gonad. However abundance of Hc-fau transcripts decreases significantly in diapausing L4 of H. contortus.3. Regulation of Hc-FAV in C. elegans developmentIn C. elegans, knockdown of Ce-rps30 confers extended lifespan, increased lipid storage in the intestine and shortened body length. These morphological characteristics are comparable to dauer larva of C. elegans, while gonad is condensed considerably. In contrast, shortened lifespan is observed in C. elegans overexpressing Hc-faua, especially in Hc-faub, where hatching failure is detected. The genes of insulin/IGF-1 signaling (IIS), TGF-P, cGMP, dafachronic acid (DA), apoptosis (AP) and fatty acids metabolism (FA) are all down-regulated in Ce-rps30RNAi worms, except for akt-1 and daf-16. However daf-16 up-regulation is inconsistent with its target genes down-regulation and the result from heat stress assay in these worms. Daf-16 RNAi conducted in Ce-rps30 (tm6034/nt1) mutants failed to rescue the worms. The S30 domain stays in the nucleus, while UBiL accumulates in the cytoplasm. Compared with Hc-FAUA, results of UBiL domain and S30 domain overexpression indicate synergism between UBiL and S30 in regulating lifespan and reproduction. These results suggest the potential functions of Hc-FAU in regulating diapause.4. Reseaching on the natural fusion protein Hc-FAUHc-FAU is a natural fusion protein, consisting of UBiL and S30, which will be cleaved in cytoplasm. In this study, we analyzed the expression of Hc-FAU in HEK293 cell line. Then western blot was performed to determine the cleavage. That showed that Hc-FAU A fusion and cleavage were both in cell, and Hc-FAUB was almost the fusion. UBiL and S30 may both conjugate to some other proteins. Homology modeling was conducted to model Hc-FAU structure. Hc-FAUB has lost 23 aminal acids in N-terminal. This may inhibit its exit nuclear. The NLS and nucleolus location were alson determined.In all, Hc-FAU may play an important role in L4 diapause. Research on the diapause mechnasm will provide a new perspective for preventing and controlling this disease. |
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