| As an obligate blood-sucking ectoparasite in arthropods,ticks can transmit a lot of pathogens including bacteria,viruses,protozoa and so on,and they are the second largest vetor after mosquitoes in the world.Haemaphysalis longicornis belongs to the Haemaphysalis genus of Ixodes,which is widely distributed in many countries around the world and it is one of the dominant tick species in China,Haemaphysalis longicornis are also widely distributed in 17 provinces of China such as Shanxi and other provinces.Haemaphysalis longicornis can transmit Theileria orientalis and Babesia ovata that have a great effect on the development of stock farming,and the SFTSV is transmitted by them can cause human severe fever with thrombocytopenia syndrome,which has a high fatality rate and can be seriously harmful to human health.miRNA is a size of about 22 nt non-coding single small RNA,it is functional through interaction with the target gene in the transcription level,the miRNA can regulate 30% ～ 90% of the genes in the body,and can participate in a variety of biological functions,such as circadian rhythm,the regulation of growth,tumorigenesis,immune response,insulin secretion,neurotransmitter synthesis and so on.Therefore,the study of the regulatory functions of miRNA in Haemaphysalis longicornis can help to lay a reliable molecular basis for the understanding of the growth and development in Haemaphysalis longicornis,and the mechanism of disease transmission,of course,it can provide certain reference data for tick-borne disease prevention and control.This study obtained miR-276-3p that may be associated with growth of Haemaphysalis longicornis by screening high-through put database of miRNA,then miR-276-3p exist were verified really in Haemaphysalis longicornis by amplifying in vitro,and detected the expression of miR-276-3p is higher in eggs,ovaries and midgut by q PCR,then that miR-276-3p may be associated with the growth and development of Haemaphysalis longicornis was speculated.In the inhibition test of miR-276-3p,the expression of miR-276-3p was inhibited successfully in Haemaphysalis longicornis after micro injecting miR-276-3p antagomir to unfed adult Haemaphysalis longicornis,then the blood sucking time,engorged weight,eggs weight and spawning time were counted,the results showed that the blood sucking time and spawning time were no significant change than the negative control group,but the engorged weight and eggs weight were significantly lower than the negative control group.It confirms that miR-276-3p can affect the fed adult engorged weight and eggs weight of Haemaphysalis longicornis.The software RNAhybrid 2.1 was preliminarily judged that e EF2 was a target gene of miR-276-3p.Then e EF2 exist really in Haemaphysalis longicornis was verified by amplifying in vitro,and the expression of e EF2 detected is higher in eggs,ovaries,malpighian tube and midgut,and the expression of e EF2 in engorged ticks is lower than it in unengorged ticks,then that e EF2 may be associated with laying eggs or sucking blood of Haemaphysalis longicornis was speculated.Then the result of the dual luciferase reporting system further confirms that e EF2 is a target gene of miR-276-3p.Through the RNAi test of Haemaphysalis longicornis,and that the inhibition of e EF2 had no effect on the blood feeding time and spawning time of Haemaphysalis longicornis was found,but it could cause the decrease of the engorged body weight and eggs weight in Haemaphysalis longicornis adults.Therefore,the results showed that miR-276-3p can interacts with e EF2,miR-276-3p and e EF2 also played important regulatory function in the growth and development of Haemaphysalis longicornis. |
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