Pale Culex Mirna Expression And Its Relationship With The Resistance Research

The mosquito-borne diseases including malaria, West Nile fever are importantinfectious diseases in the world and caused serious threat to human health. Mosquitocontrol is the main measure to combat mosquito-borne diseases. Chemical control,because of its killing speed, long-lasting and convenient features, has been the mainmethod in the comprehensive management strategy. Unfortunately, excessive andcontinuous usage of insecticides induced the development and spread of resistance.Then insecticide resistance exacerbated the spread of mosquito-borne diseases.However, after a very long time, chemical control for its rapid and efficientadvantage will still be the main method in control of mosquito-borne diseases.Insecticide resistance mechanisms include three main areas: target resistance,that is, the change of the insecticide target sites. Metabolic resistance, that is, thechange of metabolic enzymes transcription level involved in metabolic resistance,and physiological resistance, such as cuticle protein and chitin enzyme also involvedin insecticide resistance. Preliminary studies concerned more about the change ofmetabolic enzyme activity, and there is no report on gene transcription regulation. Inthis study, we wanted to explore the resistance mechanism on regulate transcriptionlevel.As an important factor on post-transcriptional regulation, miRNA was widelyfound in animals and plants. miRNA can regulate nearly30%genes throughdegradating mRNA expression or inhibiting protein synthesis. Organism’s growth,development, and apoptosis can be regulated by miRNA. Recently, there were moreattentions on miRNA against infection in mosquito. So far there is no report on therelationship between miRNA and insecticide resistance.This study selected Culex pipiens pallens (Cx. pipiens pallens) as the researchobject which is widely distributed in the northern China. We compared miRNA expression differences of deltamethrin-susceptible and–resistant strains by solexasequence. PCR and T-A cloning were used to verify pre-miRNA. Quantitative PCRfurther validated miRNA expression differences between resistant and susceptiblestrains. The dual fluorescence report test was used to verify miR-71can regulateCYP325BG3expression.In this study, the main results are as follows:1Two experimental strains were established in laboratory with WHO larvaebioassay method. LC50of the resistant strains is0.85mg/L and LC50of sensitivestrains is0.03mg/L. There is28.3times difference between the two strains.2Solexa was used to sequence the miRNA of deltamethrin resistant andsusceptible strains. mRNA of the sensitive strain sample has the concentration670ug/ml and OD260/0D280is1.80. The resistant strain sample has the concentration670ug/ml and OD260/0D280is1.83. After detected with agarose gel electrophoresis andAgilent Bianalyzer, RNA integrity is good for solexa sequencing, and the ratio ofrRNA is less than40%, suggesting that solexa sequencing results can be used tocontinue analysis.3After removing joints of the adaptors, pollution sequence and reads with lowquality from the raw data, we statisted sRNA length distribution and the sequence ofthe20-23nt length occupied high proportion through the distribution. The obtainedsequences (clean sequence) were annotated in accordance with rRNAetc> knownmiRNA> repeat> exon> intron. The miRNA precursor (pre-miRNA) of Cx. pipienspallens was extracted according to sequence of the genome of Cx. quinquefasciatusand the secondary structure was analysed. miRAlign software was further applied inthe above sequences and the sequences were aligned in miRBase.85conservedmiRNAs were identified and they have91precursors.143new miRNAs werescreened and they have209precursors in Cx. pipiens pallens. At the same time, we compared the miRNA difference between sensitive and resistant strains of Cx.pipiens pallens.4We detected insect specific miRNAs, miR-932, miR-980, miR-998, miR-999and mosquito-specific miRNAs, miR-1891, miR-1889and miR-2941in our data.miRNA cluster was analysed with the10K bp as the maximum distance. Sevenclusters were found in our data, including let-7and miR-125cluster which is mostcommon in insects. PCR and T-A cloning were used to detect the pre-miRNA. Thesequencing results show that the stem of precursor is conserved, while the nucleotidemutation is more common in the loop compared with Cx. quinquefasciatus. Thisphenomenon provides a new feasibility for the systematic classification ofCx.pipiens pallens and Cx.quinquefascias.5We selected significant different miRNA detected by solexa sequencing andfunction known miRNA for quantitative verification. The expression levels ofmiR-279-3p, miR-13, miR-989, miR-4448、miR-71and miR-285are higher in thesusceptible strain than in the resistant strain, and those of miR-317, miR-2b andmiR-92a are higher in the resistant strain than in the susceptible strain.We find the interations of miR-71and CYP325BG33’UTR sequence throughtarget prediction principle. Dual fluorescence report test confirmed the interactionbetween miR-71and CYP325BG3. We speculate that miR-71may involve indeltamethrin resistance through regulating the expression level of CYP325BG3.This study firstly reported the miRNA expression profiling in Cx. pipienspallens, identificated the differentially expressed miRNA in deltamethrin resistantand susceptible strains, and found miR-71can regulate CYP325BG3transcription.Our study has important theoretical significance and potential practical value.