Study On Differential Expression Profiles And SNP Screening Of Cold-related Genes In Paralichthys Olivaceus

The olive flounder (Paralichthys olivaceus) is an important marine economicfatfsh in aquaculture with a broad thermal tolerance from14to23°C. Under thecondition of artificial breeding, we have to elevate the temperature of the seawaterto maintain their normal growth in winter. So, how to improve the low-temperaturetolerance of flounder caused our attention. This study aimed to screen cold-tolerantrelated genes in flounder by RNA-seq, clone and analyze the expression patterns ofmajor cold-related genes, and select cold-related SNP of them, which may clarifythe mechanisms of response to cold stress and provide the molecular basis ofcold-tolerate flounder breeding in the future.The cold stress conditions were determined according to the median lethaltemperature experiment results.The cold-tolerance (CT) and cold-sensitive (CS)groups were obtained, and group without cold streess as a control. The results ofhistological structure of flounder tissues showed that the low temperature treatmentcould cause flounder gill damage, such as epidermal cells separating from theirblood vessels and swelling of the ends of gill filaments, and increase the gapbetween muscle fibers. The activity of pyruvate kinase was significantly higher inthe CT group than that in the CS group under low temperature (P<0.05). The ATPcontent in the CT group was higher than those in the CS and control groupssignificantly (P<0.05), while ATP concentration in the CS group was significantlylower than those in the control and CT groups (P<0.05).The transcriptional response of flounder to cold stress was characterized byusing RNA-seq technique. Transcriptome sequencing was carried out usingIllumina Miseq platform for CT, CS and control, and a total of29021unigenes wasgenerated. There were410genes that were up-regulated and255genes that weredown-regulated in the CT group. The numbers of gene in the CS group that wasup-regulated and down-regulated were593and289, respectively. GO and KEGGanalyses revealed that Signaling transduction, Lipid metabolism, Protein digestive,Signaling Molecules and Interaction were the most highly enriched pathways for DE genes under cold stress.CIRP, HMGB1and APO-AIV genes, cold-tolerance related genes, were selectedto be cloned in flounder using5’and3’ RACE methods. The cDNA sequence offlounder CIRP, HMGB1and APO-AIV were932bp (GenBank accession number,KC735176),1222bp (GenBank accession number, KC422724) and1063bp(GenBank accession number, KC735177), respectively. The RT-PCR expressionpatterns of those three genes in flounder tissues showed that CIRP and HMGB1wereall expressed in brain, gill, heart, muscle, liver and intestines, but the expression levelof HMGB1was lower in gill. APO-AIV has a distinct tissue-specific expression,mainly expressed in liver and intestines. Under the low water temperature, expressionlevels of CIRP, HMGB1and APO-AIV genes in flounder brain, liver and muscletissues were also studied using real time RT-PCR method. In muscle, the expressionlevels of CIRP and HMGB1increased during0~12h, and then decreased with theprolonging of treatment time. The expression level of HMGB1in brain tissueincreased during0~2h, and then decreased to the initial level, and there was nodifference at expression levels in the liver tissue during the whole cold treatment time.The expression levels of CIRP in liver tissue increased during0~2h, and thendecreased to the initial level. But there was no obvious fluctuation in brain tissue.APO-AIV expression levels in brain and muscle were increased to the highest at6hand12h, respectively, and then decreased. While in liver, the expression levelsdecreased firstly, and then slowly increased. According to above results, we speculatethat all these genes could participate in flounder cold toleration and may promoteinflammatory responses under low temperature, so they could be candidate genes forcold-resistant molecular marker selection of flounder.Furthermore, we conducted SNP screening from CIRP and HMGB1genes usingSanger sequencing and found6SNPs (4of CIRP and2of HMGB1) have significantdifferences (P<0.05) between CT and CS groups, which can be used for breeding thecold-tolerated flounder.