Study On Mitochondrial Genome Of Three Conus Species Native To Hainan

Various toxins from cone snails' venom(conotoxins,conopeptides)for predation and self defense are a huge reservoir of marine medicines.Conotoxins can specifically target on ion channels or membrane receptors,which have been used in pharmacology,pathology and neurobiology research.They are the focus of development of new drugs currently,which have received much attention in recent years.To date,there are about 900 Conus species all over the world.Some species are on the verge of extinction due to worsening marine ecological environment.It is an important method for Conus species molecular classification to use mitochondrial DNA(mtDNA)to identify the species and research their genetics and evolution.So far,there are only 9 species'complete mitochondrion genome data recorded in the NCBI Reference Sequence Database(RefSeq).Complete mitochondrion genome investigations of cone snailsnative to Hainan have not been reported yet.In this study,the mitochondrial genome DNAs of three Hainan Conus species,i.e.the C.litteratus Linnaeus,C..marmoreus Linnaeus and C.virgo Linnaeus,were extracted.Their complete mtDNAs were sequenced respectively.Molecular classification and genetic evolution analyses were performed based on these mtDNA sequences.Three methods,including improved high-salt precipitation,method,organelle/magnetic beads method and kit extraction were used to extract cone snail's mtDNA with three kinds of tissues of foot muscle,venom gland and hepatopancreas separately.The mtDNA isolation methods were optimized.It was discovered that the improved high-salt precipitation method was the best.High purity and yield of mtDNA was obtained using improved high salt precipitation way.Five pairs of primers were designed based on the conserved part of the full-length sequence of the known species' mtDNA.The mtDNA isolated by improved high salt precipitation method was used as a template to do PCR amplification.The PCR products were cloned,sequenced and jointed.The full-length sequences of the three species' mtDNA were obtained successfully.,.Their mtDNA lengths were 15 577 bp for Conus litteratus,15 579 bp for Conus marmoreus,and 15 594 bp for Conrs virgo.Each complete mtDNA contains 37 genes in total,in which there were 13 protein-encoding genes,22 tRNA genes and 2 rRNA genes.The mtDNA sequences of the three Conus species were analyzed systematically,including genome size,structure and composition.Three kinds of data sets of each Conus species were used for phylogenetic analysis,which included full-length mitochondrial DNA sequence,13 mitochondrial protein-encoding genes,and 2 rRNA genes.Genetic evolution trees for different Conus species were constructed.A analysis was established and obtaining Six phylogenetic trees with similar topology structure were obtained through NJ and ML method analyses.The results provides theoretical knowledge and a new idea for Conus mtDNA genome elucidation and their composition and function investigation,as well as molecular classification and genetic evolution analysis etc.