The Regulation Mechanisms Of MiR-275 In The Eggs Development Of Rhipicephalus Sanguineus

Rhipicephalus sanguineus is a kind of absorption blood parasite,parasitizing in mammals,birds,reptiles,is also the media of bacteria,viruses and other pathogens,seriously effects human health and livestock production.Recently,the emergence of drug resistance and environmental pollution problems,the traditional method of drug control tick has been gradually replaced by a biological control tick,such as Bm86 and Bm95 vaccine were successed in commerce.This means that it is important of screening analysis tick molecular function.MicroRNA is a kind of endogenous non-coding RNA,only 18～25 nt,many studies shown that miRNA widely exist in plant and animal cells.These special molecules combined with target gene mRNA specifically,to inhibit synthesis of the protein and induction degradation of the mRNA,thus take part in the regulation of the gene expression.MicroRNA induce gene silencing,has been confirmed that it involved in the regulatory process of cell growth,development,gene transcription and translation,and many other life activities.In this study,we selected miR-275 in the database,which was related to egg development and researched the regulatory mechanism of miR-275.To predicte target genes using miR-275 regulated target gene promoter transcription regulatory regions and validate its authenticity using RNA interference and Real time PCR(qRT-PCR).We cloned target gene by polymerase chain reaction(PCR),analyzed its sequence,constructed recombinant expression vector using the function domain of target gene,it was expressed in vitro and its immune response was studied.MiR-275 is an important factor of regulating gene,it is great significance to analyze its expression level in different development stage.We analyzed the mechanism of miR-275 regulated target genes by RNA interference,qRT-PCR and animal experiment.The results showed that vitellogenin gene(Vg)is an important target gene of miR-275.Designed specific primers referencing vitellogenin(registration number:EU086096)nucleotide sequence,got a gene which was 1218 bp.The genes was high homology with Rhipicephalus microplus,up to 93%,then was 85%,78%and 62%with D.variabilis、Amblyomma hebraeum and Ixodes scapularis respectively.The Vg gene contained a Von Willebrand Factor tyeD(VWFD)domain which had 221 amino acids.we designed primers of the specific function which been expressed in E.coli,the recombinant protein molecular weight was 51 kDu,it showed reactivity in different level with some positive serum such as rabbit anti-Rhipicephalus sanguineus,rabbit anti-Haemaphysalis longicornis,rabbit anti-Hyaloma detritum and rabbit anti-Dermacentor nuttalli.The results showed that the relationship between miR-275 and Vg gene regulated negtive by qRT-PCR from the different the sucking ticks in Rhipicephalus sanguineus,while during the zero to five days and nine to thirteen days showed that regulated positive when five to nine days regulated negtive.RNA interference showed that cause egg development disorder,such as spawning quantity reduce,hatching rate low,hatching time extension,egg shape anomaly,etc.Noteworthily,when Rhipicephalus microplus sucked blood for long time the Vg gene expression was reduced and miR-275 expression was increased gradually.This result showed miR-275 was involved in the regulation Rhipicephalus microplus suck the blood and was a negative regulatory role.The results indicate that miR-275 takes part in the eggs development and blood digestion in the Rhipicephalus sanguineus.When the expression level of miR-275 is gradually increased,the expression level of Vg is decreased during the eggs development.In one respect of blood digestion,because of the expression of miR-275 has been in high expression levels,when the Rhipicephalus sanguineus start sucking,with the help of the miR-275,the expression of Vg gene began to gradually increase and involved in the digestion of blood.The results can also be verified by RNA interference experiments of Vg gene.Furthermore,miR-275 is a negative regulatory mechanism in the middle of eggs development and blood digestion of Rhipicephalus sanguineus,while during the early days and the later period showed the regulating positive.The study fill the function of microRNA regulation of ticks blank at home and abroad.At the same time,in order to detail analytical study of the mechanism of its regulation lay the foundation,for the development of biological control measures ticks provides a theoretical basis.