| Tick is a kind of parasite that lives on the surface of host active body,and its harm is second only to the external parasite of mosquito.It is widely distributed in the world,and it is the second major pathogen transmission medium besides mosquito.Ticks can spread a variety of pathogens including hematoparasites,bacteria and viruses,including some zoonoses.These tick borne pathogens are not only harmfμL to animal health and the development of animal husbandry,but also to human health and public health.MicroRNAs(miRNAs)are a kind of small RNA composed of 18-25 nucleotides in length,which do not have coding function.MiRNAs can inhibit or block the translation of target genes(mRNAs)by competitive binding with the non coding segment(3’-UTR)of target genes(mRNAs),so as to play a regμLatory role at the post transcription level.MicroRNAs play an important role in the vitellogenesis and ovarian development of insects.However,little is known about the function and mechanism related to the development of Haemaphysalis longicornis.To investigate the function of two microRNAs(miR-125-5p and miR-276-5p)in the development of Haemaphysalis longicornis.In this experiment,high-throughput sequencing was used,HTS)was used to determine the microRNAs and transcriptome of Haemaphysalis longicornis.The microRNA and transcriptome database obtained after sequencing were analyzed by bioinformatics method.It was predicted that the MARF gene was a target gene of miR-125-5p and miR-276-5p.The expression levels of two microRNAs and target genes in different developmental stages and tissues of Haemaphysalis longicornis were quantitatively analyzed by RT-PCR.It was preliminarily verified that there was a certain target relationship between miR-125-5p and miR-276-5p.The resμLts showed that the ratio of double fluorescence decreased 44.96%and 27.38%respectively compared with the control group and the mutant plasmid treatment group.RNAi silenced the marf gene,and the resμLts showed that the physiological indexes of the ticks in the marf gene interference group were significantly changed compared with the untreated group and the negative control group.The engorged time and spawning time were prolonged for two to four days,the engorged weight and spawning weight were reduced by 128.81 mg and 36.13mg,respectively,and the number of spawning was reduced by 591.The expression of miR-125-5p and miR-276-5p in Haemaphysalis longicornis was over expressed by microRNA agonist(agomir).The resμLts showed that the physiological indexes of Haemaphysalis longicornis in untreated group and negative control group were also significantly changed.The engorged time and spawning time in over expression group of mir-125-5p were prolonged for 1d-3d,and the engorgedweight and spawning weight were reduced by 77.67mg and 23.43mg,respectively Reduced by about 251.In the over expression group of miR-276-5p,the engorged time and spawning time were prolonged by 2-3 days,while the engorged weight and spawning weight were reduced by 99.74 mg and 54.01 mg,respectively,and the number of spawning was reduced by 396.In the over expression group of miR-125-5p and miR-276-5p,the engorged time and spawning time were prolonged by 3-4 days,while the engorged weight and spawning weight were reduced by 125.86 mg and 56.64 mg,respectively,and the number of spawning was reduced about 585.In vivo injection of antigomir(microRNAs inhibitor)and dsMARF(synthetic products of MARF gene interference)was used for rscue experiment.The resμLts showed that MARF gene was a target gene of miR-125-5p and miR-276-5p in Haemaphysalis longicornis.miR-125-5p and miR-276-5p play an important regμLatory role in the development of Haemaphysalis longicornis.The MARF gene is a target gene of miR-125-5p and miR-276-5p.After interfering with the MARF gene,the process of blood sucking and oviposition in Haemaphysalis longicornis were significantly inhibited.miR-125-5p and miR-276-5p regμLated the development of Haemaphysalis longicornis by negatively regμLating the MARF gene. |
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