Culex Pipiens Pallens RNA-seq And Relationship Study Of CPGs Among Resistance

Mosquitoes transmit a lot of pathogens,causing a series of diseases such as malaria,dengue fever,West Nile fever,Japanese encephalitis and filariasis,which became a severe threat to public health worldwide.Therefore,it is imperative for controlling and preventing mosquito-borne diseases.However,due to the lack of effective vaccines and special effects drugs,mosquitoes controlling relying on insecticides became a main strategy.And one of strategy is chemical control,because of its fast killing and convenient characteristics.Unfortunately,continuous and irregular application of insecticides has led to a constant increasing resistance,which became an outstanding problem of great concern to the general public.As we all known,mosquito resistance mechanisms to insecticides included metabolic resistance,target resistance,cuticle resistance and behavior resistance.Both are mainly involved in gene amplification,gene expression difference,gene mutation.Now,there are many researches in metabolic resistance and target resistance home and abroad,but less in the cuticle resistance and behavior resistance.The cuticle of mosquito covers its entire surface,play a role of protecting its form in development,and defending against external disadvantage.There is no doubt that cuticle immensely strengthen the viability and adaptability of mosquitoes.It ensure that mosquitoes evolved into one of the most successfully groups in the world.The cuticle has been hypothesized to play a role in insecticide resistance by reducing penetration or sequestering insecticide.We selected different physiological development stage of Culex pipiens pallens,they are: first?second?third?fourth instar larvae?pupal and femal mosquitoes who become adult mosquitoes for three days,and has not suck blood.After the total mRNA extraction from them,we used new generation of high-throughput sequencing technology,the platform is Illumina Hiseq,screening for the genes of the cuticle protein associated with drug resistance throughout the genome,the significant Differentially Expressed Genes?DEGs?of log2 Fold change>3.0 and Padj<0.05 were selected,and real-time fluorescence quantitative PCR was used to verify the DEGs.Subsequently,the dsRNA genes of the target genes were injected into the mosquito to verify the effect of objective genes on the susceptibility of cypermethrin.Main results for this study are as follows:1.We sequenced 6 samples used Illumina Hiseq platform,on average we generated about 6.66 Gb bases from each sample.After mapping sequenced reads to reference genome and reconstruct transcripts,we finally get 13,517 novel transcripts from all samples,of this,8,653 are previously unknown splicing event for known gene,665 are novel coding transcripts without any known features,and the remaining 4,199 are long noncoding RNA.2.Differentially Expressed Genes?DEGs?: With gene expression result,we detected DEGs between samples,among them,the up regulated genes are 1035,944,657 in each sample;and the down regulated genes are 2680,1215,975 in each sample.Finally,between samples,we found 167 up regulated genes and 145 down regulated genes.3.Gene Ontology Analysis of DEGs: With DEGs,we perform GO classification and functional enrichment for DEGs.GO has three ontologies: molecular function,cellular component and biological process,we would perform functional enrichment respectively.The molecular function include 224 genes,cellular component include 149 genes,and biological process include 272 genes.4.Objective genes validation: selected two DEGs from comparison among groups,quantitative real-time PCR analysis showed that,gene XM₀01845883.1 in resistance strain expressed highly than sensitive strain,suggest that cuticle protein gene accelerate to the form of resistance.In conclusion,our study reported the results of RNA-seq between Culex pipiens pallens resistance strain and sensitive strain,and verified three cuticle protein genes,one of them up regulated led to the forming of cuticle resistance,which provided a reference for the formation reason of mosquito cuticle resistance,in addition,maybe provide a new perspect to control diseases vector.