| Ticks are a kind of ectoparasites in vertebrate hosts, which are the vectors of various kinds of diseases. The innate immune response is the key link between tick and pathogen. But so far, there is little information to understand the mechanisms of tick innate immune. Micro RNAs(mi RNAs) are 18-25 nt non-coding small RNAs that regulate target messenger RNAs at the post-transcriptional level. Mi RNAs combine with 3’-UTR of target m RNAs to repress their expression or degrade the target m RNA, and then play an important role in biological regulation at post-transcriptional levels, such as growth development and innate immunity etc. In order to study the role of mi RNAs in tick innate immunity, basis of existing database of differentially expressed mi RNAs in the laboratory, this paper injected lipopolysaccharide(LPS)-innate immune stimulant of the arthropods-to Rhipicephalus haemaphysaloides and identified several mi RNAs associated with tick innate immunity to provide some references for clarifying mechanism of tick innate immunity.In the present study, there are the following four contents:1 5 conserved mi RNAs LPS-induced were identified by real-time PCR, which included mi R-184, mi R-79-3p, mi R-71-5p, mi R-1-3p, and mi R-133-3p, and the down-regulation changes were 4.2、3.6、3. 4、3.3 and 5.0 after LPS induction, respectively. Moreover, the conserved mi RNAs precursor sequences were cloned by Reverse-transcription PCR, and the precursor structure of five mi RNAs were predicted by m Fold, which further validated these five mi RNAs in this kind of ticks..2 These mi RNAs had stage-specific and organ-specific expression characteristic by real-time quantitative PCR. Compared to other stages and organs, all of these mi RNAs were the most highly expressed in larvae and the fat body of adult female ticks.3 The expression of mi R-133-3p in the semi-engorged state of the fat body, salivary gland and ovaries was significantly down-regulated when compared to the organs of unfed ticks, and the down-regulation fold changes were 13.3, 4.2, 15.4, respectively. Although the expression of mi R-133-3p both significantly up-regulated in the experimental groups of semi-engorged ticks and fat body, there were no differences in the all biological indicators of blood feeding compared to control groups. However, the expression of mi R-133-3p decreased significantly in the engorged larvae infected with Babesia microti.4 The predicated target genes of mi R-133-3p and mi R-79-3p were validated by Dual Luciferase Reporter Assay System. It was found that the average relative fluorescence activity of mi R-133-3p agomirna-injected experimental group was 0.2, while the control group was 2.5. It showed the relative fluorescence activity was significantly different by T-test analysis, which indicated predicated Calcitonin gene related peptideⅠreceptor(CGRPR) was one of the target genes of mi R-133-3p. It was also found that the average relative fluorescence activity of mi R-79-3p agomirna-injected experimental group was 2.8, while the control group was 2.8. It showed there was no significant difference by T-test analysis, which indicated Splicing factor, proline- and glutamine-rich(SFPG) was not the target gene of mi R-79-3p. |
PDF Full Text Request