Study On Mitochondrial Genome And Transcriptome Of Gryllotalpa Orientialis

Mole crickets are one of the main underground pests, which is harmful to crops and gardening, belonging to gryllotalpidae, Ensifera, Orthoptera. There are about64species that have been reported in the world wide,10species of which occur in China. At present, the research about mole crickets is mainly focused on the aspects of biological characteristics, taxonomy, morphology and anatomy, pest control, the mitochondrial genome. However, studies about transcriptome and studies combined transcriptome with mitochondrial genome of Gryllotalpa orientalis have not been reported.In this paper, the specimen of Gorientalis collected from the northern of Henan province of China, was used for mtDNA and transcriptomic study. The complete mitochondrial genome was obtained using Sanger-Method Sequencing with L-PCR and Sub-PCR amplification strategies, the transcriptome data was obtaied using high-throughput sequencing technologies based on HiSeqTM2000platform of Illumina. For the mtDNA we did a comparative analysis combined with G.orientalis from Korea studied by Kim. For the differentially expressed genes that we selected are either related to gender differentiation or related to species differentiation, we have do a preliminary comparative analysis combined with the transcriptome sequence of Teleogryllus emma and Ruspolia lineosa. The main conclusions of this study are as follows:The complete mitochondrial genome of Gorientalis is15514bp in length. The mtDNA contain a standard content of13protein-coding genes (PCGs),22transfer RNA genes (tRNAs),2ribosomal RNA genes (rRNAs)and an A+T-rich region. All the protein-coding genes are starting with a typical initiation codon ATN, not exactly the same as Kim’s study. Eight of the13PCGs use the usual TAA as termination codons, whereas the remaining five genes (CO II, ND3, ND5, ND4, CytB) have incomplete termination codon (T). With the exception of the tRNASer(AGN) whose DHU arm is absent, the remaining21tRNA genes can form a typical clover leaf structure. A total of25unmatched base paires occure in the studied specimen, which is fewer than that of Kim’s results; most mismatches are G-U pairs, which form a weak bond, and the position of the unmatched base paires are also different from Kim’s results. But what is similar to Kim’s study is that all anticodon of tRNAs are identical to Drosophila yakuba and L.migratoria. The nucleotide compositions of the Gorientalis both in Kim’s Study and in this study are significantly biased toward adenine and thymine. All the three specimens present moderately decline, while the AT-skew of the Gorientalis in this study slightly higher than the other two. The genetic distance among three species based on mitochondrial genome showed that the mitochondrial genome difference of Gorientalis that living in China and South Korea had reached species level, needs further research on the taxonomic position of these species.The transcriptomic data obtained using Pair-end sequencing strategy based on high-throughput sequencing technologies was assembled into Unigene using de novo strategy, and obtained43,516Unigene of female adult,39,913Unigene of male adult, and38,166Unigene of Gorientalis (all-Unigene). Annotated38,166Unigene by Blastx revealed22,193blast hits in the Nr database,18,733blast hits in the Swiss-Prot database,16,304blast hits in the KEGG database,7,991blast hits in the COG database, and11,948blast hits in the GO database. In total,22,991Unigene had hit in above database, accounting for60.24%of total Unigene. By using blastx and ESTScan, we perdicted the reading frame of Unigene and obtained24,91CDS sequence. We also had identified2,388SSRs of the species, and had detected88,887SNPs in female and80,448SNPS in male.The analysis of differentially expressed genes (DEGs) between male and female releaved that the DEGs in the G. orientalis is the lowest among the three species (Gorientalis, Teleogryllus emma and Ruspolia lineosa), while the Teleogryllus emma is the higest. And the analysis of differentially expressed genes between different species is highter than that between male and female. The analysis of differentially expressed genes between male and female in the Gorientalis showed that20of the blast hits in molecular function ontology,9of the blast hits in biological process ontology and11of the blast hits in the cellular component ontology are function enriched in the GO database. In the KEGG Pathway enrichment analysis there are nine of112matched KEGG pathways are significant enrichment, followed by metabolism of riboflavin, legionella disease, cardiac muscle contraction, ribosomes, Parkinson’s disease, oxidative phosphorylation, tyrosine metabolism, cysteine and methionine metabolism, PPAR signaling pathways.Transcription mitochondrial genome mapping analysis results show that although the adult male and female expression of mitochondrial genome in general is consistent, but the mitochondrial transcription activity of female adult is higher than that of male adult. The COIII expression efficiency is the highest of the13PCGs, while ATP8and ND6are the lowest. LrRNA biased toward3’ while srRNA biased toward5’. The coverage of lrRNA is higher than that of srRNA, which may be caused by RNA-seq technology. There are five primary transcripts in the reads coverage map, among them2located on the J chain and3is located on a chain of N chain.