Transcriptome And Mitochondrial Genome Of Ruspoils Lineosa

Ruspoils lineosa, a popular species which belongs to Conocephalidae of Tettigonioidea in Orthoptera, is widely distributed in China. At present, the researches about Ruspoils lineosa mainly focus on the studies of the morphology and taxonomy. However, the research about mitochondrial genome and transcriptome of Ruspoils lineosa has not been reported. In order to assemble tree of life of Ensifera insects and carry out the researches about mitochondrial genome, transcriptome and molecular mechanism of individual growth and sex differentiation of Ensifera insects, we chose Ruspoils lineosa as the object of the study. In this study, the strategies of combined long-PCR and sub-PCR amplification were used to sequence the whole mitochondrial genomes of Ruspoils lineosa. Besides, the transcriptome of Ruspoils lineosa (including female and male adults) had been sequenced by Illumina HiSeqTM2000high-throughput sequencing platform. After processing the raw sequencing data, the clean data were assembled and annotated, and conducted global bioinformatic analyses. Meanwhile, the differential expression genes between male and female adults of Ruspoils lineosa were screened, and found out the differential expressed Unigene related to the individual growth or sex differentiation. Then we had analysed related functions of Unigene and identified metabolic and signal transduction pathway which the Unigene involved in. Finally, we constructed and analysed the mitochondrial genome transcriptional map using transcriptomic data. The main conclusions of this study are listed as follows:(1) The results of mitochondrial genome research showed that the length of Ruspoils lineosa’s whole mitochondrial genome was15,231bp, and the A+T content of mitochondrial genome was71%. Most of the start codons of protein-coding genes were typical triplet codons ATN, and most of the stop codon was TAA. The length of the22tRNA genes located in64～74bp. Besides, tRNASer-AGN AGN was lack of DHU arm, the remaining21tRNA were able to form a typical structure of clover. The length of LrRNA and SrRNA was1,325bp and885bp respectively. The secondary structure of LrRNA contained6structure domains and47stems; SrRNA contained3structure domains and33stems. AT riched region located between SrRNA and tRNAIle genes, with290bp in length and76.2%of AT content. The phylogenetic tree showed that Ruspoils lineosa, Ruspolia dubia and Conocephalus maculatus always clustered together, which was consistent with the traditional taxonomic results.(2) The results of the transcriptome research showed that male and female Ruspoils lineosa adults obtained54,968,554and53,645,136clean reads respectively, the GC content of male and female adults were44.75%and45.85%respectively. Among them, the female adult obtained147,380contigs, the total length was35,181,115bp with N50was299; the male adult obtained162,240contigs, the total length was38,282,436bp with N50was296, and ultimately obtained61,779All-Unigene. In addition, the research obtained26,240CDS sequences. Comprehensive annotation results for Nr, Nt, Swiss-Prot, KEGG, COG, GO were22,934,8,504,16,915,14,854,6,598,8,478respectively.24,025Unigene obtained protein function annotation, accounted for38.89%of All-Unigene. Functional classification results showed that6,598Unigenes which were annotated to the COG database were involved in25function classes.8,478Unigene which were annotated to the GO database involved in three ontology (genetic molecular function, cellular components, biological processes involved in) and gained10,823,18,557,33,206annotations respectively, and the functional category of three ontology was16,18and23respectively.14,854Unigene which were annotated to the KEGG database participated in258pathways. According to the classification principle of KEGG,258pathways could be divided into6types. Among the22,934Unigene which were annotated to the Nr database, the proportion of Unigene whose matching degree of annotation reached more than80%were about11.9%. In addition,47,203SNPs had been detected in the female adult, and49,651SNPs had been detected in the male adult. At the same time,2,684SSR had been detected in the study, of which the highest amount was trimer SSR.(3) The analysis results of differentially expressed Unigene showed that the number of Ruspoils lineosa’s differential expression genes were9,937. The number of richly expressed genes in male was3,820. The richly expressed genes in female were6,167. The number of differentially expressed genes which were annotated to GO database was1,348, accounting for13.50%. The richly expressed Unigene which were annotated to the GO database involved in three ontology (genetic molecular function, cellular components, biological processes involved in) and respectively gained24,26,24entries. The number of differentially expressed genes which were annotated to KEGG Pathway database was2,320, and the number of entries which were matched to Pathway was245.(4) The results of mitochondrial transcriptome mapping analysis showed that14,458sites of Ruspoils lineosa’s mitochondrial genome gained reads coverage, and the fraction of coverage was94.92%. Among them, the mitochondrial genome’s bases cover degree in Ruspoils lineosa male adult was higher than the female adult. Among the protein-coding genes, the expression level of COI was the highest in the male and female adults and the lowest was ND6and ATP8respectively. In addition, the reads coverage of J chain’s encoding gene was often higher than N chain’s, which showed that both of J chain and N chain had multiple transcription starting points. The rRNA reads coverage of Ruspoils lineosa male adult’s mitochondrial transcriptome was higher than female adult’s. Moreover, the reads cover degree of LrRNA was much higher than SrRNA’s. Among them, the average coverage of LrRNA’s base sites were28,558, The average coverage of SrRNA’s base sites were2,572, which suggests that large subunit played a more important role in the assembling of ribosome.As an important part of the National Natural Fund Project "the comparison of orthoptera insects and evolutionary mitochondrial transcriptome studies" which was hosted by professor Yuan Huang, this topic was the first study about Ruspoils lineosa mitochondrial genome and transcriptome, which could provide another important transcriptome information for Tettigoniodea insect’s research.