Transcriptome Analysis Of Liver And Head Kidney In Rainbow Trout (Oncorhynchus Mykiss) Reveals The Response To Heat Stress By RNA-seq

Fish are ectotherms and usually exposed to daily or seasonal temperature fluctuations in their aquatic environments.Although fish can cope with some ranges of temperature variations through physiological plasticity or micro-evolution,severe diseases and death occur when they exceed their thermal tolerance capability.Mean water temperatures are increasing across most of the globe given the growing threat of global warming,which is inevitably challenge to farmed fish in semi-open aquaculture,especially cold-water fish.Therefore,physiological responses and thermal plasticity have become the focus of current stress research.The rainbow trout is an economically important fish in the world.The limited stress tolerance of this species to high summer-like temperatures usually leads to mass mortality and great economic loss.However,there is limited information on the mechanisms underlying moderate heat responses in the rainbow trout.In this present paper,using female fish belonged to full-sibling inbreeding families as experimental materials,the transcriptomes of two tissues(liver and heat kidney)of rainbow trout mild stressed at temperatures up to 24 ? were profiled and compared with those of the fish living at a normal temperature of 18 ? by using the Hiseq? 4000 sequencing platform;meanwhile,the genes expression results of the RNA-seq data were validated by the RT-q PCR.The main results obtained as follows:1.More than 277 million clean reads were obtained from 6 liver libraries and aligned against the rainbow trout genome.Of 27796 obtained genes,1452 genes were only expressed in control group(OM_CL),2332 genes were only expressed in heat stress group(OM_HL),and 24,012 genes were co-expressed in both groups.A total of 128 unique transcripts were differentially expressed in the liver under heat stress and control conditions with a cutoff of q-value<0.05,many heat shock protein genes for thermoregulation such as HSP47,HSP90,and HSP70 were identified,some novel genes involved in heat stress like CYPLA3,CCT3,and PDIA3 were also found.Nine of the differently expressed genes were further validated by RT-q PCR which confirmed the high reliability of the RNA-seq results.Gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analyses revealed that several pathways,including those for protein metabolism,energy metabolism,and immune system,were influenced by heat stress.Moreover,an important protein-processing pathway in the endoplasmic reticulum(ER)was identified,and the key role of ER-associated degradation(ERAD)and function of calpain as an upstream regulator of apoptosis were confirmed under heat stress.2.After trimming and filtering the raw reads,281 million clean reads with 42.45 Gb total read length were generated from six head kidney libraries.In total,40,055 genes were obtained from the control group(OM_CHK)and heat stress group(OM_HHK)after mapping to the rainbow trout genome.A total of 36,271 genes co-expressed in both groups;1534 and 1547 genes were only expressed in OM_CHK and OM_HHK groups,respectively.With q-value<0.05 as the threshold,a total of 443 differentially expressed genes(DEGs)were detected between the OM_CHK and OM_HHK groups.Among these DEGs,125 were novel genes;236 genes were downregulated and 207 were upregulated.These included one or more members from HSP90,HSP70,HSP60 and HSP40 families and some cofactors or cochaperones.RT-q PCR validation suggested the high reliability of the RNA-seq results.GO and KEGG analysis were conducted to gain insight into physiological and gene pathways.Specifically,genes involved in protein folding,unfolded protein binding,and mutiple biological processes involved in the maintenance of an internal steady state or adapting regulation from an initial condition to a later condition were highly induced.“Protein processing in ER”,“Phagosome”,“NOD-like receptor signaling pathway” and “Spliceosome” pathway were enriched.Protein processing in ER which belongs to Genetic Information Processing was also the most significantly enriched pathway in the head kidney of rainbow trout,with 38 genes upregulated and 1 gene downregulated.In the ER,protein restricted to a quality control system that ensures correct folding or eventual degradation of misfolded polypeptides by ERAD.Two pathways involved in regulation of immune system,namely,“Phagosome pathway” and “NOD-like receptor signaling pathway”,which strengthens immune,apoptotic and inflammatory responses,helping to survive cells.3.By comparing the transcriptome data of head kidney and liver of rainbow trout under heat stress,it was found that 36 common differentially expressed genes in this two tissues which mainly included HSP coding genes;the protein processing in ER and “NOD-like receptor signaling pathway” which be related to immune system were significantly affected by heat stress in the two tissues.In conclusion,the results of this study provide a comprehensive overview of heat stress-induced transcriptional patterns in rainbow trout liver and head kidney,which would be particularly useful for further studies on the molecular mechanisms underlying responses to heat stress in this species and extend the potential for identifying genes that may be critical for adaptation to changing climates.