Dugesia Japonica (dugesia Japonica) Mitochondrial Proteome And Transcriptome Studies

Dugesia japonica is well-known specie in Planaria (Platyhelminthes:Turbellaria: Tricladida). Turbellaria have a special position in metazoan evolutionary history. It is the first animal that appeared mesoblast and bilateral symmetry. Dugesia japonica is an important experimental material in research of regeneration, embryonic development and cell differentiation because it's unique structure and strong regeneration properties.Mitochondria play an important role in producing energy, transforming material and cell apoptosis. It is responsible for more than90%of all energy in cells and many physiology and pathology process. The structure and function's alter of mitochondria is related to more than100kinds of human diseases, such as the degenerative disease, Down's syndrome, diabetes, heart disease, aging and cancer. It is also a key factor of cell apoptosis. All of these have closely relationship of mitochondrial genome, transcriptome and proteome. It is not only one way flow from the transcriptome to proteome in gene expression, but both are connected to each other. To understand the regulatory role of transcriptome and proteome, we need research RNA and protein expression in mitochondria at the same time.We used density gradient ultra-centrifugation to obtain high pure mitochondria, then its mitochondrial proteome were investigated using Shotgun (SDS-PAGE electrophoresis and nano-LC+ESI-MS/MS analysis) techniques and mitochondrial transcriptome were analyzed using Solexa (RNAseq) techniques. The results are as follows:1. Mitochondrial proteome were analyzed using Shotgun (SDS-PAGE electrophoresis and nano-LC+ESI-MS/MS) techniques and data-dependent MS/MS spectra were obtained simultaneously. MS/MS spectra were automatically searched against the Swiss-port database and Mitpromic mitochondrial database using the BioworksBrowser rev.3.1, and we also reference mitochondrial protein data about C. elegans and Drosophila.307proteins are identified in total. The DAVID Bioinformatics Resources6.7was used to GO annotation, the results showed that215proteins (70.36%) are located in mitochondria,76proteins(24.9%) are unknown function,15proteins are located in intracellular. There involved TCA and oxidative phosphorylation in energy metabolism; mitochondrial transporter; signal molecule and mitochondrial genome replication, transcription, translation, protein. Using SubMito1.1KEGG pathway metabolic pathway analysis,247of which is located as mitochondrial protein (80.46%), relates to oxidative phosphorylation, TCA and mitochondrial encoding related protein;32functions of unknown proteins (10.42%); the28proteins (9.12%) located within the intracellular or other cell organelles.2. The mitochondrial transcriptome in Dugesia japonica reveals by Solexa deep RNA sequencing (RNA-seq) with total1.18Gb data, and assembled into54,364unigenes. Using Blast X to farther annotate, total22457unigenes (41.31%) are notes. GO classification analysis showed that13025unigenes are correlated to ten functions. Compared the GO results of mitochondrial proteome and transcriptome, we can observes that proteins of gene expression and genome maintenance are major component, and it is24.9%in mitochondrial proteome,43.16%in mitochondrial transcriptome. KEGG pathway annotation identified7395unigenes.3. Using directional deep sequencing and ZOOM software, we aligned1.12million sequenced reads uniquely to the Dugesia japonica mitochondrial genome, finding almost the entire mitochondrial genome to be transcribed (94%J strands and88%N strands). Furthermore, less than4.5%of the mitochondrial genome sequence was represented singly within the sequenced libraries, indicating the near-exhaustive depth of this sequencing., we obtainned6primary transcript segments and9%gaps through the transcriptome mapping.Mitochondrial genome of Dugesia japonica includes11PCGs (atp8and ND4L deletion),22tRNA genes,2rRNA genes. It cannot entirely matched from mitochondrial transcriptome to genome.4. Comparing GO results between mitochondrial proteome and mitochondrial transcriptome, we can concluded that the proteins of cell apoptosis is remain stable in both data, at around1.6%. It shows the factors of apoptosis were stable in posttranscriptional processing and transportation. We presumed thatthese factors of apoptosis had special protection mechanism during posttranscriptional processing and transportation.5. The correlational comparison of mitochondrial proteome and transcriptomeThe comparison of two "-ome" used13025unigenes in transcriptome and230proteins in proteome, the data were deal with Box-Cox algorithm and normality processing. The spearman correlation coefficient were calculated and the result were spearman r=0.92. It explained the230proteins in the transcription and translation level correlation is better.Through comparing the expression information between transcriptome and proteome, we analyse differences of gene expression level in Dugesia japonica mitochondria, and reveals the regularity of gene expression and post-regulation. We wish to explore the special expression information in Dugesia japonica mitochondria. At the same time, these information about mitochondrial proteome and transcriptome in Dugesia japonica will provide research foundation to platyhelminth.