| Tick is blood-sucking ectoparasite that acts as a vector to transmit a variety of pathogens.The life history of tick includes four stages: eggs,larvae,nymphs,and adults.As with many media,ticks,mainly through the bite of a host transfer of pathogens.Because of ticks characters of blood sucking and strong reproductive ability,there is currently no specific drug for the prevention and control of it.The new control strategies to control the growth and reproduction of tick can be a key measure to prevent ticks,in which basic research in molecular biology is essential.The breeding of tick is a key link in prevention and control.The vitellogenin is the main component of the eggs.Studies have shown that vitellogenin(Vg)is transferred through clathrin-mediated endocytosis and then digested by enzymes to form Vitellin(Vn),but the specific mechanism is unclear.Vg is a storage protein regulated by juvenile hormone and ecdysone in ticks.Vg is also mediated by vitellogenin receptor(VgR),As Vn stored in the cytoplasm,it provides material and energy for oocyte development,which is very important for the development of oocytes.Clathrin with a molecular weight of 180 kDa consists of its unique "triskelion" and participates in a variety of cell biology processes,including the growth,reproduction and development of species.Its endocytosis plays a crucial role in nutrient absorption and pathogen invasion.Therefore,this study identified for the first time the heavy chain molecules of the clathrin of ticks and observed its function in the process of Vn production.1.Molecular cloning and sequence analysis of Rh-ChcThe open reading frame(ORF)of Rh-Chc is 5103 bp and encodes 1670 amino acids(aa)with 7 CLH domains,a theoretical isoelectric point of 5.52,and fat coefficient of 94.61.The index is 38.83 indicating it is a stable protein,and its averagehydrophilicity is-0.221,which is a hydrophilic protein.Phylogenetic analysis with the amino acid sequence of Rh-Chc indicated that the evolution of RH-Chc was closest to that of Ixodes scapularis,followed by Stegodyphus mimosarum.2.Prokaryotic expression of Rh-Chc and preparation of polyclonal antibodyAccording to the epitope prediction,the amino acid sequence of 978-1269 was selected.Rh-Chc gene was cloned and used for the construction of Rh-Chc/pET30 a expression plasmid,which showed that 827 bp fragment had been inserted.After recombinant plasmids were transfected with Escherichia coli Transetta(DE3),the recombinant plasmid expressed Rh-Chc peptide with the protein size of 28 kDa in the form of inclusion bodies under the induction of 1.0 mM isopropylthiogalactoside(IPTG)at 25°C for 16 h.,.Under this condition,a large amount of the protein of interest was obtained.After dialysis and purification using urea as a denaturing agent,a high-purity protein of interest was obtained,and a rabbit anti-Rh-Chc polyclonal antibody was prepared using the protein.In addition,screening peptides by epitope prediction(DRAYEFAERCNEPGVWSQL)was coupled with KLH and prepared mouse anti-Rh-Chc polyclonal antibody,Western Blot analysis showed that the Rh-Chc polyclonal antibody specifically binds to Rh-Chc protein,The protein was specifically bound in the ovary at the fifth day of blood-sucking obtained by microscopically dissection.Immunofluorescence results showed that the rabbit anti-Rh-Chc polyclonal antibody had better specificity in the ovary at day 3 of fed adult females.3.Distribution of Rh-Chc in organization and stagesThe quantitative Real-Time PCR(qRT-PCR)technique was used to analyze the Rh-Chc gene in the different stages of the R.haemaphysaloides: eggs,unfed larvae,fed larvae,engorged larvae,unfed nymphs,fed nymphs,unfed adult females,fed adult females,and organs including ovary,fat body,salivary gland,and midgut were also studied from different feeding adults(unfed adult females,fed adult females and engorged adult females).The results showed that Rh-Chc was expressed widely at 8developmental stages and in 4 tissues of 3 feeding adults.In the detection of 8developmental stages,it was found that higher expression was observed in fed larvae and unfed adults,but the lowest in the unfed nymph.In the detection of 4 tissues of feeding adults,it was found that the expression in the fat body was higher in the unfed stage,followed by that in the midfield and salivary glands.In the fed and engorgedstages,the expression is higher in the ovary,midgut,salivary glands and fat bodies in sequence.Immunofluorescence was performed in the ovary,salivary gland,and midgut on day 3 of fed adult females obtained by microdissection,which was found that the protein expression level in the salivary gland was higher,followed by that in the ovary,and the expression of the midgut was the lowest.4.Expression of Rh-Chc and Rh-Vg after RNA interferenceThe Rh-Chc gene was silenced by dsRNA-mediated RNA interference,and the Rh-Chc gene knockdown was analyzed by qRT-PCR.After the microinjection of dsRNA in experiment group with the concentration of 0.4 ?g ds Chc,0.8 ?g ds Chc and1.2 ?g ds Chc,most of the ticks were feeding on the rabbit.With the concentration increasing,the upper body rate was gradually reduced,the amount of ticks dropped from rabbit at day 3 of feeding.Compared to the control group(injected with dsRNA Luciferase),injection of different concentrations of ds Chc affected tick growth and inhibited tick engorgement.The size of ticks was smaller than that of the control group,and there was no egg spawning in the Chc dsRNA group.The tick engorgement rate of control group was 90% and the spawning rate was 100%.Rh-Chc and Rh-Vg were detected by qRT-PCR technique on day 3 of feeding after RNA interference.The transcription level of Rh-Chc was decreased after interference,and the transcription levels of Rh-Vg1,Rh-Vg2 and Rh-Vg4 was decreased significantly.Compared with the control group,the transcription level of Rh-Vg3 had no difference by microinjection of 0.4 ?g ds Chc and was significantly reduced with increasing concentration(microinjection of 0.8 ?g ds Chc,1.2 ?g ds Chc)..5.Functional study in the process of Vn productionRNA interference was used to silence the Rh-Chc gene by in vivo microinjection and in vitro ovarian culture,the dsRNA was injected into the adult tick(female),and the ovaries were collected at day 5 of feeding after 48 h,96 h and 144 h to perform immunofluorescence,.The results showed that the development of oocytes was significantly slowed down,and the expression level of Rh-Vg was also reduced after the silencing of Rh-Chc.Adult tick(female)ovary was obtained by microdissection on day3 of feeding,day 5 of feeding,day 1 of engorgement,day 3 of engorgement,day 5 of engorgement,day 7 of engorgement,and then placed in L-15 medium adding Rh-Chc dsRNA.It was found that the expression level of Rh-Vg in the ovary at day 1 ofengorgement was increased.In other stages,the expression level of Rh-Vg was decreased.The results of dsRNA interference were consistent with the results of microinjection and addition of endocytic pathway inhibitor Pitstop-2 in L-15 medium.It was indicated that during the process of exogenous and endogenous Rh-Vn synthesis,the Rh-Vn production in the ovary of silenced ticks is reduced.It was also found that Rh-Chc and Rh-VgR colocalized on the cell membrane of the oocyte at day 3 of feeding,which was speculated that Rh-Vg may be mediated by Rh-VgR and Rh-Chc.In summary,Rh-Chc played an important role in the growth and reproduction of tick,It participated in the formation of Rh-Vn and the development of the ovary.Due to the character of Rh-Chc,it can be used as potential drug targets to provide the reference of preventing and controlling ticks and tick-borne disease research in future. |
PDF Full Text Request