The Inhibition And Killing Effects Of Rhipilin-1 Gene Double-stranded RNA On Rhipicephalus Haemaphysaloides

Ticks is a common kind of ectoparasites in humans and animals as haemophilus arthropods.In the taxonomy,the ticks belongs to Arthropoda,Arachnids,Acarina.It has four developmental stages which including spawn,larvae,nymph,adult.Ticks is a variety of zoonosis pathogen reservoir host and media,transmitting and infecting Tick-borne encephalitis,protozoosis,Xinjiang hemorrhagic fever,relapsing fever,Lyme disease,Q fever,threatening public health and doing harm to livestock economy.Therefore,it is important to prevent and control the spread of ticks.Rhipicephalus haemaphysaloides is a kind of hard ticks,which widely distributed in the south of the China.In currently,the main method to control ticks is using chemical drugs,and it takes serious environmental pollution caused great harm to the host though the killing ticks effect is good.With the development of biotechnology research,the new strategy of prevention and control methods gradually formed in ticks.RNA interference has been widely used in modern ticks researches.Our laboratory has cloned and identified Rhipicephalus haemaphysaloides anticoagulant molecule Rhipilin-1.And it has been proved that RNA interference can be effectively reduced capacity of ticks blood sucking by micro injection.This study was based on the principle of RNA interference.Directly soaked dsRNA on the surface of ticks.By the way of soaking with lipid agent,aim to achieve RNA interference effect.So as to implement the new control techniques such as tick pesticides.Details are as follows:1)The recombinant plasmid pGEM-T-Rhipilin-1 and pGEM-T-Luciferase as template,according to T7 kit instructions,synthetic double-stranded RNA of Rhipilin-1 gene and Luciferase gene.2)Screen the best conditions during the soaking process,such as lipid agents,soaking time and soaking proporation.Detect the labelled dsRNA distribution by the observation of Cy3 fluorescently.3)Experiment on animals with the best optimal lipid agent,soaking time,and soaking proportion in every stage.The results showed that the best soaking conditions were as follows:larvae lipid agent Lipofectamine 2000,soaking time 24h,double-stranded RNA:lipid agent with a volume ratio of 2:1;nymphs lipid agent DMRIE-C,soaking time 12h,double-stranded RNA:lipid agent with a volume ratio of 1:1;Adult ticks lipid agent DMRIE-C,soaking time 12h,double-stranded RNA:lipid agent with a volume ratio of 1:2;After soaking,the ticks engorged time increased and engorged weight reduced significantly,which have a significant impact on the ticks original biological function.In short,the Rhipilin-1 gene could be a candidate gene,which for RNA interference methodology of tick prevention and control practices.This study provide a reference in the creation of new applications and environmental insecticide.