| Ticks are ectoparasites that can parasitic on the body surface of a variety of animals and suck blood.During suck blood,they destroy the tissues and skin of the host,and at the same time spread many diseases which endanger the health of humans and animals.Therefore,the prevention and control of ticks is of great significance in the fields of animal husbandry and public health.Nowadays,chemical insecticides are mainly used to prevent and control ticks.However,chemical insecticides are harmful to the environment and can easily cause ticks to develop resistance.Therefore,there is an urgent need to develop low-cost and environmentally friendly resistance.Tick preparations.The ecdysone receptor(EcR)of arthropods is of great significance for regulating their normal growth and development.The previous studies has indicated when the expression of the EcR gene of Rhipicephalus haemaphysaloides is disturbed,it can seriously affect its blood sucking and moulting.Biological characteristics such as spawning.In order to screen the target molecules to control tick and evaluate their application potential,this study wad made that the cloning of the EcR genes of two other common species of ticks,and used double-stranded RNA with synthetic EcR sequences for anti-tick research.In this study,amplification primers were designed based on the EcR sequences,total RNA of Hl and Ha salivary glands was extracted,and constructed the salivary gland c DNA library of Haemaphysalis Iongicornis and Hyalomma asiaticum.c DNA was then synthesized,and Hl-EcR and Ha-EcR genes were amplified by PCR technology.;The amplified products were respectively connected with the pGEX-4T-1 plasmid vector to construct a recombinant plasmid,and the recombinant plasmid was identified by PCR and sequencing,and its sequence was analyzed.The results showed that the total length of Hl-EcR ORF sequence was 1263 bp,the total length of Ha-EcR ORF sequence was 1260 bp,and the homology of Hl-EcR with Rh-EcR and Ha-EcR sequences was 87.4% and 87.5%,respectively.It shows that the EcR gene is highly conserved.The 657bp-1197 bp of Hl-EcR was selected as the dsRNA sequence,the size of this fragment was about 540 bp,and its homology with Rh-EcR and Ha-EcR was 90.6%and 90.2%,respectively.After synthesize dsRNA,and conducting of RNA interference to Rh with it,Hl and Ha ticks were microinjected,their interference effects was evulate by fluorescence quantitative PCR,and the biological behavior of ticks after interference also was investigated.The results showed that the m RNA expression levels of the EcR genes of the three ticks,Rh,Hl and Ha after interference were reduced by 89.6%,93.9% and 87.0% respectively,and the saturation rate and egg laying rate were both 0%.Chitosan nanoparticles were synthesized by reverse microemulsion method and their characterization was verified.The microscopic morphology of the chitosan nanoparticles is spherical with good dispersibility;the particle size is 143.7±23.5 nm,and the particle size distribution is uniform;the surface potential is 36.9±0.5 mV;the combination with dsRNA is ideal;the maximum drug loading capacity CS:dsRNA(m:m)=24:1;it can protect dsRNA from Ribonuclease A degradation.After using CS-dsRNA(dsRNA=2 μg/μL)to infect the ticks for 48 h,the EcR expression and biological characteristics of the adult ticks of the three species were not significantly affected;The expression of tick EcR was significantly reduced(p<0.05),and the rate of satiety and molting were both 0%.This conclusion found that the usage of conserved sequences of ds EcR can interfere with the EcR of different ticks,and all of them can seriously affect their biological behavior,which preliminarily clarifies that the EcR gene has the potential function against tick.The use of CS-ds EcR has almost no effect on adult ticks,but it can seriously affect the biological behavior of the ticks,and make them unable to complete their life history normally.It will provide a new means for the research,prevention and control of ticks. |
PDF Full Text Request