| Haemonchus contortus is a gastrointestinal parasitic nematode that causes major economic losses resulting from hemorrhagic gastritis,anemia,edema and associated complications in small ruminants.As there is no effective vaccine available,treatment of parasitic nematode infections relies heavily on anthelmintic.However,the widespread use of these anthelmintic has resulted in serious resistance and drug residue problems worldwide.Therefore,it is imperative to develop new intervention strategies.One of the possibilities is the rational design of anti-parasite drugs and/or vaccines,built on the deep understanding of the biological and developmental processes in these parasites.A lack of effective genetic and genomic tools for some parasitic nematodes and an inability to maintain their entire life cycle in vitro hampers detailed functional studies.Fortunately,Dauer hypothesis paved the way for parasitic nematode research,providing a model nematode as a surrogate system.The Dauer hypothesis proposed that L3 larvae of parasitic nematodes and Dauer larvae of the free-living nematode Caenorhabditis elegans may employ same molecular mechanisms in regulation their development based on their similarities in morphology,behavior and developmental process.Four signaling pathways including cyclic guanosine monophosphate signaling pathway,Dauer transforming growth factor ? pathway,insulin like signaling pathway and steroid hormone pathway regulates Dauer formation in C.elegans,thus it's speculate that Dauer transforming growth factor ? pathway and insulin like signaling pathway also regulates L3 arrest in parasitic nematodes,and block of two pathways either,the L3 may failed to further develop.However,this hypothesis needs to be verified.In the present study,three genes(Hc-daf-3 and Hc-daf-5 in TGF-? pathway and Hc-akt-1 in insulin-like signaling pathway)were isolated from H.contortus by molecular method and analyzed by bioinformatics technology.Their transcriptional levels were also accessed by qPCR.Transgenic technology was used to study the expression patterns in C.elegans N2 strain.Specific polyclonal antibodies against synthetic peptides and recombinant protein were produced to detect the localization of Hc-DAF-3 and Hc-DAF-5 in H.contortus.AKT inhibitor IV and RNAi method were used to uncover the importance of three genes in the transition from free-living to parasitic stages.In addition,BiFc was also performed to verify the interaction between Hc-DAF-3 and Hc-DAF-5.(1)The structural and functional characterization of protein kinase B encoding gene(Hc-akt-1)The full-length genomic DNA sequence of Hc-akt-1 was 10431 bp.Hc-akt-1 encodes two transcripts,Hc-akt-1a and Hc-akt-1b,which were 1644 bp and 1659 bp respectively.Hc-AKT-la and Hc-AKT-1b were Ser/Thr kinases,containing conserved Pleckstrin Homolgy Domain and Kianse Domain.Homology modeling revealed that the difference between Hc-AKT-la and Hc-AKT-1b lies in the aD helix of kinase domain.Phylogenetic analyses showed its close relationship with Ce-akt-1.Hc-akt-1a and Hc-akt-1b were transcribed in all stages including Egg,L1,L2,L3,L4F,L4M,AF and AM,while the transcription level of Hc-akt-1a was highest in adult stage,and that of Hc-akt-1b was highest in L3.Compare with Hc-akt-1b,Hc-akt-1a was up-regulated in egg and adult.Transgenesis in C.elegans showed that Hc-akt-1 was expressed in ASJ neurons in N2 strain of C.elegans.Silencing Hc-akt-1a and Hc-akt-1b simultaneously in C.elegans exhibited similar phenotypes as silencing of Ce-akt-1,including prolonged survival time and enhanced drug resistance to paraquat.Silencing both Hc-akt-la and Hc-akt-1b of H.contortus xL3 by soaking method with specific dsRNA blocked the xL3 development to L4.AKT inhibitor IV also blocked the xL3 development to L4 in vitro.(2)The structural and functional characterization of co-Smad encoding gene(Hc-daf-3)The full-length genomic DNA sequence of Hc-daf-3 was 9487 bp and had 16 exons and 15 introns.The CDS of Hc-daf-3 was 2097 bp in length.Hc-DAF-3 was a co-Smad,containing typical MH1 Domain and MH2 Domain,connected by linker region.Phylogenetic analyses showed its close relationship with hookworm,but a distint relationship with Ce-DAF-3.Hc-daf-3 was transcribed in all stages,including Egg,L1,L2,L3,L4F,L4M,AF and AM,and up-regulated in L3 and adult female.Transgenesis in C.elegans showed that Hc-daf-3 was expressed weakly in vulva cells in N2 strain of C.elegans.The immunohistochemical results indicated that Hc-DAF-3 was expressed in the reproductive organs and cuticle of adult worms.Silencing Hc-daf-3 of H.contortus xL3 by soaking method with specific siRNA blocked the xL3 development to L4,mainly the buccal development.(3)The structural and functional characterization of transcript factor encoding gene(Hc-daf-5)The full-length genomic DNA sequence of Hc-daf-5 was 8535 bp and had 11 exons and 10 introns.The CDS of Hc-daf-5 was 1629 bp in length.Hc-DAF-5 was a transcription factor,containing SDS Domain and Dach box.Phylogenetic analyses showed its closest relationship with the homologue of Nippostrongylus brasiliensis,and a closer relationship with Ce-DAF-5 compared with Hs-SKI.Hc-daf-5 was transcribed in all stages including Egg,L1,L2,L3,L4F,L4M,AF and AM,and up-regulated in L3.Transgenesis in C.elegans revealed that Hc-daf-5 was expressed in nervous system in N2 strain of C.elegans,mainly in head neurons,tail neurons and VNC.The immunohistochemical results indicated that Hc-DAF-5 was expressed in the reproductive organs and intestine of adult worms.Silencing Hc-daf-5 of H.controtus xL3 by soaking method with specific siRNA blocked the xL3 development to L4,mainly the buccal development.BiFc verified the interaction between Hc-DAF-3 and Hc-DAF-5 in vitro.Taken together,the present study explored the structure and function of two important genes from TGF-? signaling pathway and one important gene from insulin-like signaling pathway in H.contortus.This not only helps us to understand the development progress of H.contortus,particularly in the transition from free-living stage to parasitic stages,but also provides new information on Dauer hypothesis application in parasitic nematodes research.Moreover,this will laid the foundation for the development of novel drugs and/or vaccines. |
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