Sequencing And Analysis Of The Complete Mitochondrial Genome Of Chorthippus Chinensis And Mongolotettix Japonica

Mitochondrial (mt) DNA has been used extensively in studies of population structures, phylogeography, and phylogenetic relationships at different taxonomic levels, owing to maternal inheritance, a relatively rapid evolutionary rate, and lack of intermolecular genetic recombinations. In particular, complete mtDNA sequences can be informative at deep phylogenetic levels and comparisons of animal mitochondrial gene arrangements have become powerful tools for inferring phylogenetic relationships.After amplifying mtDNA using Long-PCR and Sub-PCR mrthods, the complete mitochondrial genomes of Chorthippus chinensis and Mongolotettix japonica were determined and analyzed through directly sequencing PCR products or through sequencing cloned PCR products. The organization and characteristic of the two mitogenome, including gene order, base composition, A+T-rich region, 13 protein coding genes, rRNA genes, 22 tRNA genes and their secondary structure, codon usage and amino-acid composition were surveyed. Finally, the evolution feature of mitogenomes and their phylogenic relationship were analyzed. The conclusions drawn from the study as follows.1. Mitochondrial genomes of Chorthippus chinensis and Mongolotettix japonica are all closed-circular molecules. The length of those two mtDNA is 15 599bp and 15 979bp separately, including the same 37 genes and an A+T-rich region as found in other Arthropoda. The orientation and gene order are identical to the hypothesized ancestral arthropod arrangement found in several insect orders such as Drosophila melanogaster except for two tRNA (K, D), which is same as in L. migratoria.2. Most frequently used initiation codons for 13 protein-coding genes in Chorthippus chinensis Tarb is ATG (ND2,COⅡ,COⅢ,ATP6,ND4L,ND6,Cyt b ), secondly used initiation codons is ATC (CO I,ND3,ND4), only two genes (ATP8,ND5) start with ATT, and ATA is special for ND1. In other words, all of the 13 protein-coding genes in Chorthippus chinensis Tarb have ATN for initiation codons. Except TAG termination codons for ND1 and a single T of incompleted stop codons for ND5, 11 genes have unambiguous completed termination codons TAA.3. All 22 tRNAs of Chorthippus chinensis and Mongolotettix japonica have the typical clover-leaf structure except for tRNA-Ser(AGN), which DHU arm could not form stable stem structure, tRNAs possess invariable aminoacyl stem, anticodon loop, and anticodon stem. Most of the size variation among 22 tRNAs stemmed from length variation in the TΨC and DHU arms.4. It is the remarkable difference with other insect mitogenome that Mongolotettix aponica has 360bp intergenic spacer region between tRNA^Ala and tRNA^Arg, which is not found in other orthopteroid insects. The 360bp intergenic spacer region contained 89bp repeat unit, duplicating 4 times, and encoded 30 amino-acid peptides. It maybe have some functions, such as replication origin, certain protein-coding gene and regulation element.5. Based on the combined dataset of deduced amino acid sequences of 13 protein-coding genes, using the maximum likelihood, maximum parsimony and neighbor joining methods to reconstruct the phylogenetic relationship of 7 species of orthopteroid insects. The results indicated that (Chorthippus chinensis Tarb + Mongolotettix japonica) and Locusta migratoria have closely related, forming a monophylic group with Gryllotalpa orientalis.The Chorthippus chinensis Tarb and Mongolotettix japonica belongs to Arcypteridae and Acididae respectively. To date, the mitogenomes in Arthropod have been determined for 126 species. Of 126 species has only 5 species of orthopteroid insects: 2 Orthoptera insects; Blattodea, Mantodea and Mantophasmatodea, 1 respectively. Therefore, it has a quite signification that the additional mitogenome of Orthoptera can be informative at deep phylogenetic levels and evolution.