Chinese Grasshopper Mapping Studies Comparing The Transcriptome And Mitochondrial Transcriptome

For assembling tree of life of Orthoptera insects and carrying out the research about mitochondrial transcriptome gene expression and molecular mechanism of individual growth and sex differentiation of Orthoptera insects, we constructed the cDNA library of male adult, female adult, female nymph of Acrida cinerea respectively and carried the transcriptome sequencing using the HiseqTM2000platform, and assembled the sequencing reads into Unigene using the de novo strategy, and conducted annotation of GO functional classification and KEGG pathway analysis to the Unigene. Besides, we found out the Unigene of differently expressed related to the individual growth or sex differentiation under the transcriptome comparison between the nymph-adult and female-male, and carry analysis about identification of related functions and metabolic pathway of the Unigene involved in. In addition, we conducted analysis of reads and Unigene mapping to mitochondrial DNA sequence of Acrida cinerea as reference genome. The main conclusions of the study are as follows:(1) We obtained2x34,779,159clean reads of female nymph,2x35,838,536clean reads of female adult and2x26,234,028clean reads of male adult after data filter and reached a total of6.95Gb,7.16Gb and5.24Gb trascriptome sequencing data respectively. There ware59,699Unigene assembled from original reads and reached31,938,874bp totally under the de novo and merge sample strategy. The female nymph, female adult and male adult had53,623,55,802and50,177Unigene assembled respectively under the strategy of sample clean reads map to Unigene.(2) There had26,700of59,699Unigene obtained annotation information after function annotation of gene using the blast tools, and22,176of which is annotated to Nr,17,057to the Swiss-Prot,6,770to the COG and9,295to the Nt. We obtained12,075Unigene with GO function classification information and24,645Unigene with annotation information in KEGG pathway, but more than50%Unigene had no function information. These detailed function and classification information provide essential information for the study of Life activities and gene regulation which is Acrida cinerea taken part in.(3) We extracted5,444DEGs between the female nymph and female adult sample, and8,073DEGs between the female adult and male adult sample using random sampling hypothesis testing of the value of RPKM. And, identifying229Unigene which are only expressed in female nymph,442in female adult,848only in male adult. These Unigene provide important information for the identification of Unigene which is related to the individual growth or sex differentiation.(4) The results of GO function classification and enrichment analysis of above DEGs showed that the female nymphs have more specially gene expression than female adults in metabolism, cellular component organization as well as process of the body’s normal metabolism maintaining. However the female adults have more specifically gene expression than male adults in the macromolecular components, organelles component, transduction regulation as well as cellular processes, and the male adults exhibit more specific expression of genes in the cell membrane components, electron carrier activity, enzyme activity regulations as well as regulation of reproductive process. According those results, we infer that female nymphs have more activity in metabolism and material accumulation process, and female adults have more activity than male adults in metabolism ensuring the progress of reproduction, but male adult give more investment of gene expression in reproductive and environmental adaptation. At the same time, the results of annotation and enrichment analysis of DEGs in KEGG pathway showed that there have DEGs in85of237KEGG pathway between the female nymphs and female adults,11of which is DEGs significantly enriched pathways, but between the female adults and male adults, this number is123, and20of which is DEGs significantly enriched pathways. In the Comprehensive comparison of these enriched pathways, we infer that more difference exits in metabolic pathway in Acrida cinerea growth process. But in adult stage, there is a translation of the pathway, in which more DEGs is related to reproduction and hormonal regulation pathway.(5) The results of mitochondrial transcript mapping of Acrida cinerea using clean reads of three samples showed that there is a high sequencing coverage about95.38%, and the female nymph, female adult and male adult are consistent in mitochondrial gene expression patterns and expression trends.1-rRNA, COX1and CYTB are genes with highest reads coverage.1-rRNA perhaps has a additional independent and efficient transcription promoter, the poly(A) tail shortage of s-rRNA perhaps leads to its low sequencing coverage. Some tRNA gene was obtained in the sequencing may be due to locating in transcription intermediates3’end which are usually polyadenylated, Acrida cinerea may exists five transcription units in mitochondrial transcription and processing mechanism which is similar to the Drosophila melanogaster. There probably exist9intermediate transcripts among44mitochondrial Unigene. COX1gene initiation codon should be ACC, and the incomplete termination codons of ND2, COX1, COX2, ND5may be finished by the polyadenylated during the transcript processing.This study is first time transcriptome sequencing research for species of Acrididae of Orthoptera, and the results will provide support for the construction of life tree of Orthoptera insects based Phylogenomics methods, and will have far-reaching effect on locust evolution study and phylogenetic analysis of the Orthoptera. At the same time, a large number of sex differentiation genes and individual growth-related genes, which is generated under the transcriptome comparison study, will provide help for in-depth study and analysis of locust transcriptional regulation and genetic mechanisms of sex differentiation or Individual growth.