Screening And Identitication Of Differentially Expressed Genes Of Kineococcus Radiotolerans By RNA-seq Under Radiation Stress

Kineococcus is a gram-positive,radio-resistant bacterium that was isolated from a high-level radioactive waste,it can survive in the harsh environment where contained gamma irradiation,alkali cations,salt,desiccation,high concentrations of metals,high osmotic pressure and high chemical toxicity.K.radiotolerans can be widely used in environmental protection and bioremediation as a bioresource,however,its molecular mechanisms still keep unknown.In order to investigate the differentially expressed(DE)genes of K.radiotolerans in response to radiation stress and reveal the molecular regulation mechanism and provide basic data for the research on its regulation of the overall level of life,we present the deep sequencing technology,RNA sequencing(RNA-seq)which is highly efficient and accurate,to analysis of the transcriptomes of K.radiotolerans which were cultured in the 4.5 kGy gamma radiation induced wild-type strain(RS)and the untreated wild-type strain(WS).WS and RS were selected for RNA extraction and cDNA library construction,Illumina Hiseq2000 sequencing platform was used for two samples transcriptome sequencing,using topHat software for analyzing the percentage of high-quality effective reads mapping of the genome of K.radiotolerans,using DEGseq software for calculating and analyzing the standard expression level(FPKM value)of each gene of two samples to filter out the DE genes.Using cuffdiff software for COG function analysis of DE genes,get the DE genes which were related to radiation resistance and were verified through the qRT-PCR.Due to the Illumina sequencing platform results,the raw data for the WS included 28,508,412 reads and that of the RS included 36,145,816 reads,After the quality pre-progressing,including the removal of the adapter-dimer sequences and the filtering of the low quality data,the raw data for the WS included 23,3357,920 reads and that of the RS included 29,611,930 reads.After removing the rRNA,further analysis of the clean data showed that the percentage of WS read mapping of unique locations in the K.radiotolerans reference genome was 86.15%and that for RS was 89.13%,the results met the requirements of the reliability of the sequencing.Compared to the WS,the number of total DE genes in the RS was 143,which included 63 down-regulated and 80 up-regulated genes according to the statistical analysis.The COG function analysis was used to screen the significant genes involved in the strain's response to radiation stress among the 143 genes,finding that there were 20 up-regulated genes related to radio-resistance.and the results were verified by qRT-PCR.Based on the above results,the DE analysis identified 20 genes of this bacterial strain to ionizing radiation.Of all of the 20 validated genes,the recA,ruvA and ruvB genes were considered to be the key genes in DNA damage repair,while the katA gene was found to be involved in reactive oxygen species(ROS)scavenging.This study provides the foundation to investigate the metabolic pathway and regulatory mechanism of radioresistance in k.radiotolerans.