Analysis Of The Genetic Characteristics On The Aedes Albopict Us In Different Ports Of Jiangsu Through The Application Of Mo Lecular Biology Techniques

Objective Compare the different genotypes of molecular markers by amplificating Aedes albopictus mitochondrial DNA, ribosomal DNA and nuclear DNA, in order to selecte the best suited one for Aedes albopictus identification and genetic analysis. Then conduct traceability analysis of Aedes albopictus and in vivo symbiotic Wolbachia in dozens ports of Jiangsu province,Revealing not only the distribution and genetic characteristics of Wolbachia endosymbionts in Aedes albopictus but also the relationship between the distribution of Aedes albopictus and geographical distance and the port area.Methods First, extract the Aedes albopictus DNA in the ten ports of Jiangsu Province, and amplify and sequence Col gene in the extracted mitochondrial DNA, aligning bases and constructing near France Phylogenetic tree with the results on MEGA by ClustalW Multiple alignment module. Second, connect the ribosomal ITS DNA gene into plasmid which is to be loaded into the vector by chemical conversion. Sequence the gene after cloning, then constructe phylogenetic tree and perform Mantel tested by sequencing results. Third, analyze the nuclear DNA by AFLP technique, digest the DNA with high frequency/low shear enzyme (EcoRI/MseI) and connect them by a linker primer, after pre-amplifing the ligation product with universal primers, selectively amplificate of that which is optimized by selective amplification primer.Construct "0" to "1" matrix with the result of PCR products by capillary electrophoresis, calculate The genetic similarity coefficient by NTSYS software then conducte Mantel test and build UPGMA phylogenetic tree.Perform homology analysis on Aedes albopictus of different ports of Jiangsu Province,through the AFLP analysis of the ITS rDNA genes and nuclear DNA.Results Identificate and perform homology analysis of the Aedes albopictus in Jiangsu ten ports by three methods. The first way,we can commendably identify the Aedes albopictus by mtDNA-COI gene amplification but can not distinguish the Aedes albopictus in different regions well for the reason of high homology, fewer base difference, small genetic differences by phylogenetic analysis.The second way, the phylogenetic tree which is constructed after cloning and sequencing the ITS gene of ribosomal DNA shows that gene sequence of Aedes albopictus in different ports of Jiangsu Province is divided into two branches. From the major terms, the Aedes albopictus of Lianyungang is divided into a separate.In another branch Aedes albopictus of Wuxi is separated from those including Changzhou, Taizhou, Xuzhou and Yangzhou. What’s more, Aedes albopictus of Changzhou and Taizhou are devide into further smaller branches.The third way, do AFLP analysis of the nuclear DNA, by capillary electrophoresis, screening ten primer combinations which are most suitable for AFLP analysis of Aedes albopictus. The ten optimized primer pairs are amplified into396bands including a total of31sites,365polymorphic loci. It gets the polymorphism percentage of92.2%, rich polymorphic loci and can be very good for distinguishing Aedes albopictus of different regions.Mantel test results show that there is no linear correlation between genetic distance and geographic distance of the Aedes albopictus in ten ports of Jiangsu Province.In the similarity coefficient of0.66by UPGMA phylogenetic tree, Aedes albopictus can be divided into three braches namely one is Wuxi, another is Lianyungang, other eight regions including Changzhou, Huai’an, Nanjing, Nantong, Suzhou, Taizhou, Xuzhou and Yangzhou compose one.Conclusion Experiments show that rDNA-ITS gene amplification and nuclear DNA AFLP analysis can both accomplish homology analysis of Aedes albopictus, but AFLP analysis is for whole genome which is rich polymorphism, better to reflect the genetic differences between the Aedes albopictus of different regions. The experiments also reveals that it is regional of ports rather than geographic distance that has linear correlation with geographic distance of Aedes albopictus in ten ports of Jiangsu Province laying a foundation for further research.