| Haemonchus contortus is a kind of blood sucking parasite in ruminant abomasum of cattle,sheep and camel.It is widespread in China and causes great economic losses to China’s animal husbandry.Anti-helminth drugs have good effects on the treatment of H.contortus,but in recent years,with the abuse and mixed use of antibiotics and drugs,drug-resistant strains of H.contortus appear frequently,which increases the difficulty of the disease control.Screening of key genes of nematode life regulation as new drug targets and the development of anti-helminth drugs are important research directions for haemonchosis control.Molting is a necessary life stage in the growth and development of nematodes.When the process is obstructed,it can affect the normal development of nematodes,reduce the activity,and even lead to the death of larvae.Therefore,it is very important to explore the molting mechanism and the biological function of related genes for the prevention and control of the disease.In this study,three nematode astacins(NAS)were selected from the published transcriptome database of H.contortus.The full-length sequences of Hc-nas-5,Hc-nas-31 and Hc-nas-33 were amplified,and their conserved-domain prediction and molecular evolution were performed.The expression characteristics of HcNASs were analyzed in 293T cells,Caenorhabditis elegcns and H.contortus.The functional role of Hc-nas-33 in the growth and development of H.contortus was explored by transcriptional analysis and RNA interference.The yeast cDNA library of H.contortus was constructed,and the candidate interaction proteins of Hc-NAS-33 were screened,and the interaction between Hc-NAS-33 and guanine nucleotide-binding protein subunit beta-1(Hc-GPB-1)was verified.The function of Hc-gpb-1 during molting was analyzed.The results lay a foundation for further study on the molting mechanism of H.contortus,and also provide a potential drug target for the development of new drugs.1.Analysis of the characteristics of HcNASsThe full-length DNA and mRNA sequences of three astacin-like genes Hc-nas-5,Hc-nas-31 and Hc-nas-33 were amplified and gene structure,protein function and molecular evolution were analyzed by bioinformatic softwares.The prokaryotic expression vectors were constructed to express the recombinant proteins and polyclonal antibodies were prepared.The results showed that three HcNASs all have a typical zinc metalloproteinase domain,in addition,Hc-NAS-31 has a cub domain and a SXC domain,Hc-NASs-33 also has an EGF domain and a cub domain.Hc-NAS-5 and Hc-NAS-31 have C-terminal signal peptides.The recombinant HcNASs were successfully expressed by pET prokaryotic expression system,and the polyclonal antibodies with good specificity were obtained by immunizing experimental animals.In this study,three HcNASs were amplified and analyzed by bioinformatics.Polyclonal antibodies were prepared,which can be used for subsequent analysis of protein expression characteristics.2.Expression characteristics of HcNASsIn order to analyze the expression characteristics of HcNASs,we constructed eukaryotic expression vectors and transfected into HEK 293T cells to analyze the localization of HcNASs in eukaryotic cells;heterologous expression plasmids were constructed and transgenic worms were generated by microinjection to explore the expression of HcNASs in C.elegans.Polyclonal antibodies were used to analyze the expression characteristics in H.contorlus by indirect immunofluorescence(IFA)in larva and Immunohistofluorescence(IHF)in L4 and adult.The results showed that all three HcNASs were expressed in the cytoplasm.In C.elegans,Hc-NAS-5 was only expressed in a few cells in the pharynx and tail,while Hc-NAS-31 could not be expressed,and Hc-NAS-33 was expressed in the pharynx and intestine.Hc-NAS-5 and Hc-NAS-31 were located in the epithelial syncytia of L3.In adult worms,the expression patterns of Hc-NAS-5 and Hc-NAS-31 were not specific,and they were widely distributed in a variety of tissues.Hc-NAS-33 was specifically expressed in the intestinal-muscle interface,the uterine membrane and the periphery of immature eggs.This study clarified the expression characteristics of HcNASs and pointed out the direction for the follow-up functional research.3.The functional role of Hc-nas-33 in the molting development of H.contortusIn order to explore whether Hc-nas-33 is involved in the molting process of H.contortus,the transcription characteristics of Hc-nas-33 in different periods were analyzed by quantitative real time PCR(qRT-PCR),and the homologous genes of C.elegans were compared.Secondly,RNA interference was used to explore the effect of silencing of Hc-nas-33 on the growth and development of H.contortus.The results of transcriptional analysis showed that the transcriptional level of Hc-nas-33 oscillated during the L1-L2 molting process of H.contortus,and reached the peak between the middle and late stages.Ce-nas-33 also showed oscillatory transcriptional characteristics during the molting processes.After RNA interference,the growth and development of larvae were seriously affected,and the larvae showed developmental retardation,decreased mobility,deformity and molting dysfunction.These results indicate that Hc-nas-33 is a molting related gene of H.contortus,which lays a foundation for further study on molting mechanism of H.contortus and development of new drugs.4.Construction of H.contortus yeast cDNA library and screening and identification of proteins interacting with Hc-NAS-33In order to further explore the molecular mechanism of Hc-nas-33 involved in the molting process,the cDNA library of H.contortus was constructed in this study,and the candidate proteins interacting with Hc-NAS-33 were screened by yeast two hybrid system;the relationship between Hc-NAS-33 and interacting protein was verified by pull-down,Co-immunoprecipitation and eukaryotic co-localization.Six candidate proteins interacting with Hc-NAS-33 were identified by yeast library screening;GST-Hc-NAS-33 recombinant protein was successfully expressed by insect baculovirus system,and the interaction between Hc-NAS-33 and Hc-GPB-1 was verified by pull-down,which was consistent with the results of CO-IP.Co-localization of Hc-NAS-33 and Hc-GPB-1 was also found in HEK 293T cells.This study successfully screened the interaction proteins of Hc-NAS-33,which lays a foundation for the follow-up functional study in the molting process of H.contortus,and helps to better understand the molting molecular mechanism of H.contortus.5.The mechanism of Hc-NAS-33 and Hc-GPB-1 involved in the molting processIn this study,we analyzed the co-localization of Hc-NAS-33 and Hc-GPB-1 in C.elegans by using the expression system,and studied the function of Hc-GPB-1 in the growth and development of H.contortus by RNA interference.The results showed that the Hc-NAS-33 and Hc-GPB-1 expressed in C.elegans were partially co-localized.After the silencing of Hc-gpb-1,the larvae also showed developmental retardation,decreased activity and molting dysfunction.Transmission electron microscope results showed that the thickness of new cuticle was affected after the decline of Hc-gpb-1,and gaps appeared in the tight junction between cuticle,epidermis and muscle cells and these were further verified by the qRT-PCR analysis of related genes.In this study,we found that both Hc-nas-33 and Hc-gpb-1 were involved in the synthesis of new cuticle during molting.This provides an important basis for further elucidating the molting mechanism of H.contortus,and also provides a new candidate drug target for drug research and development of H.contortus.In conclusion,three astacin genes were amplified from H.contortus,and their protein expression characteristics were confirmed by bioinformatics analysis.The results showed that Hc-nas-33 was involved in the molting process.Six candidate interaction proteins of Hc-NAS-33 were screened by yeast two hybrid system,and their interaction with Hc-GPB-1 was verified.Finally,the biological functions of Hc-NAS-33 and its interacting protein Hc-GPB-1 in the molting process were studied by heterologous expression and RNA interference. |
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