Analysis Of Differential Expression For MiRNA In Different Life Stages And The Function Of Novel MiRNA-2 In Haemaphysalis Longicornis

Haemaphysalis longicornis(H.longicornis)is an important pathogen vector,which can transmit a variety of pathogens.As a three-host tick,H.longicornis needs to go through egg,larvae,nymph and adult stages.Different developmental stages have their own physiological characteristics and are also regulated by complex gene networks.Micro RNAs(miRNAs)is a kind of small molecules with a size of about 22 nt.It is widely involved in the regulation of a variety of physiological functions,such as cell growth,differentiation,tissue growth,organ and phylogeny.Therefore,it is of great significance to explore the expression and mechanism of miRNAs in different developmental stages of H.longicornis.In this study,H.longicornis was taken as the research object,and the expression profiles of miRNA in different developmental stages of H.longicornis analyzed by miRNA were found by high-throughput sequencing technique.The specific and differentially expressed miRNAs in screening stage.GO enrichment and KEGG analysis showed that these miRNAs were involved in the regulatory pathways related to the development of H.longicornis,indicating that these miRNAs may play an important role in the corresponding developmental stages.And make an in-depth study on the regulation mechanism of novel miRNA—novel miR-2,which is the key to the regulation of its growth and development.The following results are obtained:1.Analysis of micro RNA expression profiles dynamic in different life stages of H.longicornis ticks by deep sequencing of small RNA libraries.In this study,high-throughput sequencing method was used to sequence the miRNA of four different life stages of H.longicornis(egg,unfed larvae,unfed nymph and unfed adult stages).A total of 133 miRNAs(78 conserved miRNAs and 55 potentially novel miRNAs)containing stage specific miRNAs and differentially expressed miRNAs were identified.In the four stages,miRNA-1 and miRNA-10 showed high abundance expression.MiRNA-315 accounted for 43.77% of all conservative miRNA transcripts in H.longicornis egg.MiR-307 was only expressed in low abundance in the eggs,but not in the other three life stages of H.longicornis.However,the potentially novel miRNA-2 was highly expressed in unfed larvae,unfed nymph and unfed adult stages,but not in egg.GO enrichment analysis and KEGG analysis showed that these miRNAs were involved in the regulation of tick development-related regulatory pathways,indicating that these miRNAs may play an important role in the corresponding developmental stages.2.Identification and functional verification of CPR1 gene3′ UTR and 5′ UTR of cuticular were successfully obtained by RACE technology.The complete cuticular gene was obtained after splicing.The length of the cuticular gene is 1171 bp and the coding sequence(CDs)is 339 bp(73bp-472bp).It encodes a total of 113 amino acids and is about 18 k Da in size.The results predicted by Signal P software showed that the 1-18 position of cuticular was signal peptide sequence,and the cleavage site of signal peptide was located at amino acid 17-18,without transmembrane region.The amino acid sequence contains a chitin-bind-4 conserved motif,which belongs to the CPR family.Due to it is the first CPR family protein found in ticks,and we named it CPR1.The CDs region of CPR1 was ligated to pet-30 a vector and CPR1 was expressed successfully.The rabbit specifical CPR1 polyclonal antibody was successfully prepared by immunizing New Zealand white rabbit with the CPR1 protein.The RNAi experiment of CPR1 proved that the silence of CPR1 could lead to serious molting defect of the nymph of H.longicornis,the molting rate decreased to 46.67%,and the molting time was prolonged to 21.71 ±0.3387 days.3.Identification and functional verification of novel miR-2.It’s the first time to found and identified tick-derived novel miRNA——novel miR-2.The precursor of novel miRNA-2 was determined and 62 bp in length,and the secondary structure contains the classical stem-loop structure.The mature sequence of novel miRNA-2 is 22 nt.The results of the target gene prediction showed that the novel miRNA-2 had a binding site located at the 661 bp of CPR1,which was in the 3′ UTR of CPR1 gene and conformed to the classical binding mode.The results of Double Luciferase Reporter System showed that novel miRNA-2 could bind to the 3′ UTR of CPR1,which decreased the activity of luciferase expressed in pmir GLO vector and CPR1 was proved to be the target gene of novel miRNA-2 in vitro.The results of in situ hybridization showed that CPR1 and novel miRNA-2 were co-located in the epidermis,indicating that CPR1 could interact with novel miRNA-2 in a spatially dependent manner.Overexpression of novel miR-2,could significantly reduce the expression of CPR1 protein,resulting in molting defect of H.longicornis.The results of phenotypic rescue test showed that the molting time of Ant / NC i Cut group was significantly delayed to 17.95 ±0.3104 days compared with Ant / i Cut group(14.32 ±0.0171 days),but the delay time was shorter than that of i Cut group(21.4 ±0.3625 days).The statistical results of molting rate showed that compared with Ant / NC i Cut group,the molting rate of Ant / i Cut group decreased to 76%,but higher than that of i Cut group(46.67%).To sum up,the precursor of novel miR-2 has a classical stem-loop structure,mature miRNA is 22 nt in length.And novel miR-2 is participating in the molting process of H.longicornis by regulating the expression of CPR1 gene.Therefore,we identified it as a ture miRNA in H.longicornis and named it hlo-miR-2.