COI Gene Polymorphism Analysis Of Aedes Albopictus And Preliminary Study On Cloning、Transcription Level Of AGO2and Dcr-2Genes

Mosquito is one of the normal insects in nature,they belongs to the family Culicidae,genus Diptera,Class Insecta Linnaeus,as transmission media of so many important diseases.There are three subfamilies of mosquitoes:Toxorhyncitinae、 Anophelinae and Culicinae,and among them the mosquito of Anophelinae and Culicinae which can transport pathogene to animal host, are the most important insect vectors in medical.Mosquito not only blood sucking to ruffle human beings,but also spreads many diseases that directly threaten people’s health in most parts of the world,and become the most serious problems of the public health.The pathogene which use mosquitoes as dissemination medium to transfer from one host to another, need to build an effective transmission system in insect vectors and avoid to kill them at the same time.The ability of mosquito to transmit pathogene is control by the hereditary factors of mosquito itself.Under the evolutionary selection pressure of nature,there were gene flow among the mosquito populations,as a result the population genetic structure changed,and one species may contain two or more different genetic types of populations.To study the relation of population genetic variation and the capability of diseases transmission will be especially important for prevention and control of important insect-borne infectious diseases.Use various analytical techniques of molecular biology for analyze the population genetic of mosquitoes and investigate the population genetic structure and gene flow are very important to insect-borne infectious diseases.Kamgang et al. assessed the genetic variability and population genetics of Aedes albopictus isolates in Cameroon.They collected Aedes albopictus mosquitoes from12localities and analyzed polymorphism at six microsatellite loci and two mitochondrial DNA regions (ND5and COI).The low genetic variation observed across the sampled Aedes albopictus isolates of Cameroon is in keeping with recently mutiple introduction and spread in this country.The genetic structure of natural populations points to multiple introductions from tropical regions.Mitochondrial DNA used as the marker of genetic differential analysis and distinguish the vector of specific arboviruses are commonly exist in mosquitoes.When the mosquitoes are infected by arboviruses,they actually are the process of innate immune defense in mosquitoes against pathogen infection.RNA interference (RNAi) as an innate antiviral mechanism in mosquito plays a key role in biological antiviral infection,the domination of biological development and on cell growth or apoptosis.A number of studies have shown that the innate immune response of the mosquito vector using siRNA pathway in anti-viral defense,AGO2and Dcr-2genes play a key role in RNA interference,and the revise or reorganize of the RNAi pathway should give us critical insights into virus-vector interactions leading to transmission. Vargas et al. show that since impaired the pathway of Aedes aegypti by silencing the expression of dcr2,r2d2and ago2genes which encoding important sensor and effector proteins in the RNAi pathway, DENV2increased virus replication in the vector and decreased the extrinsic incubation period required for virus transmission.In our research,we clone,identify and analyse the genetic polymorphism of mitochondrial cytochrome C oxidase subunit I gene (COI) in Aedes albopictus from Guangzhou to investigate the association between mosquito and epidemic of Dengue fever. We also perform molecular cloning of the AG02and Dcr-2gene fragments of Aedes albopictus and characterize the transcription level of these two genes across all the life stages of Aedes albpictus and before or after infected by DENV2for evaluation of the interaction between DENV and the RNAi response.Objective:1. To analyse the genetic polymorphism of mitochondrial cytochrome C oxidase subunit I gene in different populations of Aedes albopictus from Guangzhou.2. To amplify AG02and Dcr-2genes and analyze by bioinformatics analysis.3. To characterize the transcription level of these two genes across all the life stages of Aedes albpictus.4. Intrathoracic inoculation of mosquitoes by Dengue virus type2to analyze the transcription level of AGO2and Dcr-2genes before and after mosquito infection.Methods:1. Aedes albopictus mosquitoes were sampled and identify as larvae,pupae and adult in12localities in Guangzhou during September2010.2. Genomic DNA was extracted from single Aedes albopictus mosquito to amplify and clone COI gene,then purification of its PCR product.3. All the PCR products of COI gene were digested by the restriction enzyme TaqI into small fragments to selected the divergent samples by analysis of single-strand of comformation polymophisim.4. The COI gene from selected samples were ligated into pMD18-T,the plasmid then subjected to sequencing.The obtained sequences were analyzed for variable sites followed phylogenetic tree reconstruction.5. The CODEHOP primers of AG02and Dcr-2were programming designed to amplified the DNA fragments,using the cDNA of female mosquito as template,and the sequencing was analyzed by bioinformatics analysis.6. The specific primers were designed to amplify the target genes and analysis of transcription level from different developmental stages of Aedes albopictus.7. Inoculate live dengue virus in the brain of suckling mice and collecte the infected culture supernatants.8. Inoculate infected culture supernatants to the C6/36cell to propagation of virus then determine virus titer.9. Establish the method for intrathoracic inoculation of mosquitoes by virus.Prepare a number of mosquitoes infected by DENV2.10. Extract total RNA from two-day-old mosquitoes(control group) and DENV2infected mosquitoes of1to14days post-infection and amplify by RT-PCR,to analyze the relative transcription level of AG02and Dcr-2.The gray value of bands were measured using ImageJ image analysis software,with data collection and statistica analysis are carried out.Results:1. The COI gene fragments from individual mosquito of different populations were cloned and digested by restriction enzyme TaqI into3small fragments successfully.2.13samples contained various banding patterns were selected by SSCP-PCR.3. The sequences of COI from13samples are all709bp in length.The average of A+T base composition is67.42%which abide by its rules in A+T bias in insects.The alignment of these COI sequences covers altogether697conservative sites,66variable sites(1.69%),including9transition sites and3transversion sites.The range of genetic distance among COI sequences is0.000to0.007.4. Phylogenetic trees presentes genetic diversity of COI from different localities of Guangzhou,of which samples from Conghua,Panyu,Nansha,Zengcheng and Tianhe assemble into one cluster, then Luogang and Baiyun assemble into another cluster. All individuals of Aedes albopictus mosquitoes from diffrernt populations assamble into a stable monophyletic group with high support values.5. The AGO2and Dcr-2genes were amplified from Aedes albopictus by using the CODEHOP primers with326bp and491bp in length,respectively.Homology and bioinformatics analysis shows the obtained sequences from target genes.6. The transcription of AGO2and Dcr-2genes were detected in all the life stages of Aedes albopictus which the highest transcription levels of these two genes were significant difference detected in female than any other developmental stages.7. Dengue virus type2was successfully passaged in C6/36cell and undiluted virus titer was6.71log10infectious virus/0.1mL.8. The AGO2gene transcription levels has no significant difference before and after DENV2infected1to14days post-infection.The Dcr-2gene transcription levels was significantly elevated in7and14days post-infection(P<0.05vs. ctrl group),and in11days was significantly decreased(P<0.05). Conclusion:1. There are not only gene flow among different Aedes albopictus populations of Guangzhou,but also genetic variation among a portion of individuals.2. The AG02and Dcr-2fragments which are key players in RNA interference were cloned successfully and the cDNA sequences were submit to Genbank (ID: JQ764670and JQ764671)3. The AG02and Dcr-2genes were involved in the growth and development of Aedes albopictus which show an important function of RNA interference during the developmental of mosquitoes.4. The difference level of Dcr-2gene transcription in different infection times suggest the inference of a mechanism termed RNA interference effect the process of virus infection and transmission.