Clone And The Effect Development Of DsRNAS For Tickicide Of Rhipicephalus Haemaphysaloides P0 Gene

As a obligative ectoparasites, ticks mainly smite mammals, birds, reptiles and amphibians.The harm of ticks lies not only in its blood-sucking from human beings and the host animals, but also in spreading these pathogens, including viruses, bacteria and protozoa, which cause diseases in humans and animals.Currently, the main prevention of ticks is chemical pesticides, which can pollute the environment and harm food safety. New prevention contains vaccine and biological control, but they are not succeed and other new prevention need to be studied.In recent years, with the development of RNAi technology, RNAi has been used in the studies of gene function of many animals, including ticks, mosquitoes and others. In view of this, P0 gene is taken as a target gene and its double-stranded RNA are used to explore its potential to kill ticks based on the principles of RNAi in this study.In this study, P0 gene of Rhipicephalus haemaphysaloides was successfully cloned by 3’RACE and5’RACE method from cDNA.The full-length sequence of P0 gene was 1118 bp, and there was an open reading frame encoding 319 amino acids, which had not signal peptide sequence.The size of P0 protein speculated was about 35 ku.The consistency of P0 of Rhipicephalus haemaphysaloides and other tick species was high. Compared with Rhipicephalus microplus and Haemaphysalis longicornis, the homology of P0 gene was 94% and 87%, respectively. The P0 gene was highly expressed in prokaryotic expression system by p GEX-4T-1 vector.The recombinant fusion protein was nclusion bodies in the bacterial cells and the size of it was about 60 ku.P0 antiserum was prepared by mice immunized with P0 recombinant protein.The result showed that salivary proteins of Rhipicephalus haemaphysaloides could be identified.P0 dsRNA and Luciferase dsRNA were successfully synthesized using T7 RiboMAXTM Express RNAi System in vivo. The three types of transfection reagents, including Lipofectamine 2000, CellfectinⅡand DMRIE-C, respectively mixed with double-stranded RNA, which transfected into Rhipicephalus haemaphysaloides to explore the best transfection reagent, the best proportion of transfection reagent and double-stranded RNA, and the best time to dip switch for the various stages. The results showed that the optimal transfection reagents of the adult, the nymphs and the larva were DMRIE-C. For the adult and the larva, the best mixture ratio of transfection reagents and dsRNA was 1:1, while the nymphs’ was 1:2. The optimal transfer time of the adult and the nymphs was 12 h, while the larva’s is 24 h.The optimum conditions of different stages were respectively applied to miticides experiments.The study suggested that if P0 gene of Rhipicephalus haemaphysaloide was silenced, the suck-blooding relevant biological characteristics would change, such as, the decrease of attachment rate and fed body weight, elongation of the suck-blooding time, etal., and even led to death.This study confirms that there is a serious impact on the complete life cycle of ticks under the silencing of P0 gene of Rhipicephalus haemaphysaloides, and even hindered their development and led to death, which made it possible as an anti-tick vaccine candidate molecule, which is very important for ticks and tick-borne disease prevention and control.