| 研究実績の概要 |
For the fist year of the project the key experiments on profiling of expression patterns of RNA and proteins in Pv11 cell culture (obtained from embryonic cell mass of the chironomids) and tissues of the chironomids were planned. The task we successfully completed, and several set of additional experiments characterizing structure of the genome and chromosomes of the chironomid were conducted. We have selected the most promising and convenient for the analysis organs (fat body, intestine and brains) of the chironomid for whole-genome and proteomic profiling. That would suggest existing special transcription factors with activity mediated by changes in trehalose concentration in the cells or the process of desiccation itself. We further defined a number of non-coding RNA , transcribed from multi-copy conservative regions of the genome, and those with the features of enhancer RNA (i.e. non-coding RNA transcribed from enhancers in the genome) with expression patterns correlated with that of anhydrobiosis-involved genes (for examples the genes located in ARiDs region of the genome (Gusev et.al., 2014)). These RNA will be a target for in vitro experiments using siRNA on the later stage of the project.
Further, proteomic profile of Pv11 cell culture on different stages of anhydrobiosis was obtained. The stages were: wet control cells, cells after 48 h of incubation with trehalose, completely desiccated cells and the cells after 24 h of re-hydration.
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| 今後の研究の推進方策 |
According to the initial plan, cap analysis of gene expression (CAGE) provides accurate genome-wide high-throughput measurement of RNA expression focused on the determination of the transcription start site but also prediction of promoter region. The aliquots of tissues and cell total RNA samples used for mRNASeq analysis will be used. In the current project, with support of co-investigator from RIKEN Omics Center, we are going to employ construction of no-amplification non-tagging CAGE libraries for Illumina next-generation sequencers (nAnT-iCAGE, by Murata et.al, 2014), excluding commonly used PCR amplification and cleavage of restriction enzyme to eliminate any potential biases. The genome of the sleeping chironomid is small (less than 96Mbp) that will allow us to reduce cost of the experiments, as all of samples to be covered in a single run HiSeq2500 sequencing platform (Illumina).This stage will provide there main set of data:
1. Using updated genome data, the exact tissue-specific transcription start sites will be identified.
2. Data on the promoters, enhancer ncRNAs specific for anhydrobiosis in certain tissues and cell will be obtained.
3. We will identify the pool of non-coding RNA (including those missed by gene model prediction) associated with changes in gene expression upon anhydrobiosis
At the same time, data on the expression level for every above element will be obtained, providing the quantitative evidence of it activity in given tissues and cell line.
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