Molecular Charactercs And Comparative Analysis Of The Complete Mitochondrial Genome Of Lepidopteran

Lepidoptera is one of the second largest families among the insecta with more than 255 thousands described species widely distributed the throughout the world.The classification of species in Lepidoptera has been controversial and complicate.The mitochnodiral DNA(mtDNA) exists in almost all eukaryotic cells with highly copy numbers.Because of small size,compositional and nucleotide sequence conservation,maternal inheritance,and relatively rapid evolutionary rate,lack of intermolecular genetic recombination,excluding intron and intergenic spacer sequence,being easy to amplification and analysis,mtDNA has been extensively used for studying population structures and phylogenetic relationships at varios taxonomic levels.In order to enrich the Lepidoptera mitochondrial genome data,and go further into structural and evolutionary studies of the mitochondrial genomes in Lepidoptera insect,mitochondrial genomes of the two Lepidoptera species,Artogeia melete and Eriogyna pyretorum belonging to two families were sequenced,assemblyed,annotated and analyzed using the strategy of sub-PCR based on the long PCR technology.After sequence assembly and annotation,two new mitochondrial genomes combined with the thirteen other Lepidoptera species deposited in GenBank used to conduct a comprehensive comparative analysis including the base composition and codon usage of mitochondrial genome protein coding genes,secondary structure of tRNA genes,Sequence homogeneous of rRNA genes and structure of control regions,etc.Finally,the phylogenetic relationship of taxa with complete mitochondrial genome from Lepidoptera was reconstructed by concatenated amino acid sequences of 13 proteins downloaded from GenBank.Some conclusions drawn from the study as follows:1.Based on the strategy of the long PCR,a rapid and accurate approach to sequence the complete insect mitochondrial DNA was established.A set of new universal primers designed in this study can be used to amplify and sequence the mitochondrial genome from Lepidoptera,and has been successfully used to specie,A.melete and E.pyretorum.2.The size of mitcohondiral genomes of A.melete and E.pyretorum is,15140bp and 15327bp, respectively.They all have a remarkably conserved set of 37 genes and a control region known as the (A+T)-riched region.The gene order and transcription direction are the same as that of sequenced Lepidoptera species.Compared with sequenced Lepidoptera,the number and length of the integenic region and overlap region are different.The length of the intergenic regions between atp8 and atp6 is 7bp.3.The average A+T content of the A.melete and E.pyretorum mitochondrial genome protein coding sequence,rRNA and tRNA gene was corresponding well to the A+T bias generally observed in insect mitochondrial genomes.Compared the base compositon of the three codon positions of PCGs in fifteen Lepidoptera species,a common rule could be drawed as following,the first codon positions have the same T%and A%.The second codon positions all have a higher T%than A%,and the T%is as much as twice of the A%.The third codon positions have the highest A+T composition, which is nearly 90%.All the above has shown that the GC→AT evolutionary pressure is really strong.4.All protein coding genes of the A.melete and E.pyretorum start with a typical ATN codon, and the CGA start codon of the cox1 found in A.melete and E.pyretorum,the GTG start codon of the cox2 found in E.pyretorum.Majority of the 13 PCGs in these two species have a complete termination codon(TAA or TAG),while several PCGs use an incomplete termination codon T.5.The are obvious biases in the both codon and amino acid usage in Lepidoptera species mitochondrial PCGs.NNU and NNA are the most frequently used codons.Leu,Ile,Phe and Ser have the highest composition of all the amino acids.6.All the 22 tRNA genes of A.melete and E.pyretorum mitochondrial genomes have a typical cloverleaf structure except trnS1(AGN) and trn S2(UCN)which DHU arm could not form stable stem-loop structure.The length of the tRNA genes various from 60bp to 72bp.Most of mismatched base pair are G-U pairs and U-U pairs.7.The(A+T)-riched region of lepidopteran mitogenomes contains some typical structures: there is a structure including the motif 'ATAGA' and 18 to 22 bp poly-T stretch downstream of the rrnS gene that is widely conserved in lepidopteran mitogenomes,there are variable tandem repeat units in the(A+T)-riched region of sequenced lepidopteran mitogenomes except for A.yamamai, C.boisduvaliiand M.sexta,A 9-bp poly-T is found immediately upstream of trnM.8.Based on the combined dataset of nucleotide and amino acid sequences of 13 protein coding genes,using the maximum likelihood(ML) and Bayesian inference(BI) methods to reconstruct the phylogenetic relationship of fifteen species of lepidopteran insects,The resulst showed the same topologies and supported a close relationship between the Geometroidea,Noctuoidea and Bombycoidea.This result deviates from the traditional view.To further studied the phyligenetic relationships of lepidopteran insects,a larger number of insect orders and mitogenome are required.This work is the first report about the complete mitochondrial genomes on A.melete and E. pyretorum.It adds two more Lepidopteran complete mitochondrial genome sequences,and has accumulated useful information for mitochondrial phylogenomics research of insect.The general properties of the organization and structure characteristics of lepidopteran mitochondrial genome are drawed form the viewpoint of structural,comparative and evolutionary genomics through comparison of the two new genome data wjth the other Lepidopteran species data in GenBank.