| Ticks are one kind of blood-sucking ectoparasites,most of which are parasitic on mammals,a few are parasitic on birds,reptiles,and amphibians.Ticks not only consume large amounts of blood and damage the host's skin but can also spread pathogens during the biting process.They are vectors of many important diseases of humans and animals.During the tick's blood sucking process,the salivary glands are channels through which various pathogens in the ticks can be transmitted to the host.The tick`s salivary glands are crucial target organs for the prevention and control of tick-borne diseases.The salivary glands of female ticks will rapidly degenerate at the end of the tick's blood-sucking process and after engorgement.This phenomenon is mainly caused by programmed cell death mechism such as the apoptosis of salivary gland cells.In this thesis,we chose to study the inhibitor of apoptosis protein(IAP)found in the salivary gland transcriptome library and explore its role in tick salivary glands,and evalulate its application potential by RNA interference technology.The tick Rhpicephalus heamaphysaloide is a kind of dominant tick species widely found in southern China,which belongs to the hard tick family and is a three-host tick.The primers were designed based on the sequence of the salivary gland transcriptome library constructed by our lab,and the full-length Rhpicephalus heamaphysaloide IAP(RhIAP)gene sequence was successfully amplified from the salivary glands of semi-engorged adult ticks.The RhIAP ORF is a size of 1887 bp and encoding 607 amino acids.RhIAP protein is about 66 kDa in size and has three BIR domains and one RING domain.The transcription level of RhIAP in ticks at different developmental stages(eggs,larvae,nymphs,female and male),different organs(ovary,midgut,salivary gland,fat body,heamolymph),and different blood-sucking status was detected by RT-qPCR.The results showed that RhIAP widely exists in different tick development stages and in different organs.During blood sucking,the mRNA level of RhIAP in salivary glands showed a downward trend with blood sucking overall.Peptides were synthesized based on amino acid sequence and used to immunize mice to acquire the RhIAP polyclonal antibody.The native RhIAP protein in salivary glands was successfully recognized by the peptide antibody.We constructed the pGEX-4T-1/RhIAP combined expression plasmid,and the GST fusion protein was successfully induced and expressed in E.coli.Then the Rhpicephalus heamaphysaloide Caspase(RhCaspase)7,8,9 was incubated with rRhIAP.The results showed that r RhIAP can directly bind to RhCaspase7 and RhCaspase8,which suggest that RhIAP might inactivate caspase to prevent apoptosis by binding with them.The RNA interference technique was used to silence the RhIAP of salivary glands cultured in vitro.Tunnel staining showed that the degree of apoptosis in the RNAi group was significantly higher than that in the control group.At the same time,female tickts were microinjected with double-stranded RNA,and the biological characteristics of ticks were observed after being attached to the ears of rabbits.The results showed that RhIAP silencing had a great effect on ticks' blood-sucking,and the engorgement rate was 0.These results indicate that RhIAP is an important anti-tick target molecule.Combining the above results,this study identified an inhibitor of apoptosis protein in Rhpicephalus heamaphysaloide and found that it can directly bind to RhCaspase7 and RhCaspase8.Interference with synthetic double-stranded RNA will accelerate salivary gland cells apoptosis which will severely affect normal blood sucking of ticks.RhIAP is a key molecule for ticks to regulate apoptosis and plays an important role in tick salivary glands and the blood-sucking processes,which showed important application potential in tick control. |
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