State Key Laboratory of Agricultural Microbiology, Key Laboratory for the Development of Veterinary Products, Ministry of Agriculture, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, Hubei, China.
College of Animal Science and Technology, Guangxi University, Guangxi, 530004, Nanning, China.
Parasit Vectors. 2021 Oct 12;14(1):529. doi: 10.1186/s13071-021-05036-2.
Abnormal dauer formation gene (daf-5), located downstream of the DAF-7 signalling pathway, mainly functions in dauer formation and reproductive processes in the free-living nematode Caenorhabditis elegans. Although the structure and function of daf-5 have been clarified in C. elegans, they still remain totally unknown in Haemonchus contortus, a socio-economically important parasitic nematode of gastric ruminants.
A homologue of daf-5, Hc-daf-5, and its inferred product (Hc-DAF-5) in H. contortus were identified and characterized in this study. Then the transcriptional profiles of Hc-daf-5 and the anatomical expression of Hc-DAF-5 in H. contortus were studied using an integrated molecular approach. RNA interference (RNAi) was performed to explore its function in transition from the exsheathed third-stage larvae (xL3s) to the fourth-stage larvae (L4s) in vitro. Finally, the interaction between Hc-DAF-5 and Hc-DAF-3 (a co-Smad) was detected by bimolecular fluorescence complementation (BiFc) in vitro.
It was shown that Hc-DAF-5 was a member of the Sno/Ski superfamily. Hc-daf-5 was transcribed in all developmental stages of H. contortus, with significant upregulation in L3s. Native Hc-DAF-5 was localized in the reproductive organs, cuticle, and intestine via immunohistochemistry. RNAi revealed that specific small interfering RNAs (siRNAs) could retard xL3 development. In addition, the interaction between Hc-DAF-5 and Hc-DAF-3 indicated that the SDS box of Hc-DAF-5 was dispensable for the binding of Hc-DAF-5 to Hc-DAF-3, and the MH2 domain was the binding region between Hc-DAF-3 and Hc-DAF-5.
In summary, these findings show that Hc-daf-5 functions in the developmental processes of H. contortus, and this study is the first attempt to characterize the daf-5 gene in parasitic nematodes.
异常 dauer 形成基因（daf-5）位于 DAF-7 信号通路下游，主要在自由生活线虫秀丽隐杆线虫的 dauer 形成和生殖过程中发挥作用。虽然 daf-5 的结构和功能在秀丽隐杆线虫中已经阐明，但在胃寄生线虫旋毛虫 Haemonchus contortus 中仍然完全未知。
本研究鉴定并表征了 Haemonchus contortus 中的同源物 daf-5，Hc-daf-5及其推断产物（Hc-DAF-5）。然后，采用综合分子方法研究了 Hc-daf-5 的转录谱和 Hc-DAF-5 在 Haemonchus contortus 中的解剖表达。体外进行 RNA 干扰（RNAi）以探索其在 exsheathed 第三期幼虫（xL3s）到第四期幼虫（L4s）过渡过程中的功能。最后，通过体外双分子荧光互补（BiFc）检测 Hc-DAF-5 与 Hc-DAF-3（共同 Smad）之间的相互作用。
结果表明，Hc-DAF-5 是 Sno/Ski 超家族的成员。Hc-daf-5 在旋毛虫的所有发育阶段都有转录，在 L3s 中显著上调。通过免疫组织化学，天然 Hc-DAF-5 定位于生殖器官、角质层和肠。RNAi 表明，特异性小干扰 RNA（siRNA）可延缓 xL3 发育。此外，Hc-DAF-5 与 Hc-DAF-3 之间的相互作用表明，Hc-DAF-5 的 SDS 盒对于 Hc-DAF-5 与 Hc-DAF-3 的结合不是必需的，而 MH2 结构域是 Hc-DAF-3 和 Hc-DAF-5 之间的结合区域。
总之，这些发现表明 Hc-daf-5 参与了 Haemonchus contortus 的发育过程，这是首次尝试对寄生线虫的 daf-5 基因进行表征。
