Comparative analysis of transcriptome in red muscles of Thunnus albacares of different sizes
Tuna is an important marine fish with high economic value.Like other tuna species,T.albacores possesses many unique characteristics,including internalized red muscle and thunniform swimming mechanics.It maintains muscle temperature significantly above ambient water temperature using metabolically derived heat;thus,they are referred to as endothermic tunas.When tunas reach 207 mm fork length(FL),red muscle temperature can be elevated by at least 3.0 ℃ above ambient water temperature.In order to explore the molecular regulation process of Thunnus albacores growing from the poikilothermal to the endothermic,the study used RNA-Seq technology for transcriptome sequencing and bioinformatics analysis of red muscle tissues in T.albacores of differrent sizes.Experimental T.albacores were caught in the South China Sea.Three small ones(126 mm＜FL＜152 mm,30 g＜WT＜46 g)were sampled as poikilothermal group,three large ones(207 mm＜FL＜235 mm,170 g＜WT＜200 g)as endothermic group.The red muscles were then sampled.Total RNA was extracted and treated.RNA quality of every sample was assessed.The paired-end RNA-Seq library was sequenced.A total of six RNA-Seq libraries were constructed and sequenced.Datasets(clean reads)were assembled together to become one unigene library,which was matched against public databases:National Center for Biotechnology Information(NCBI)protein non-redundant(nr),Swiss-Prot,Pfam,Eukaryotic Orthologous Groups,Gene Ontology(GO),Kyoto Encyclopedia of Genes and Genomes(KEGG).The input data of DEG(differentially expressed genes)analysis was the reads count.Differential expression analysis of the three groups was performed.P-value significance threshold in multiple tests was set by false discovery rate(FDR).The fold changes of DEGs were estimated according to the read count in each sample.DEGs were selected using the following filter criteria:FDR≤0.05 and fold-change ≥ 2.Eight DEGs were randomly selected for qRT-PCR.Compared with the control group,the expression of the target gene changed exponentially and was normalized to the expression of elongation factor 1-alpha gene.A total of 146.00 and 156.88 million clean reads were obtained in poikilothermal and endothermic tuna groups,respectively.277 314 unigenes were obtained.Compared with the poikilothermal tuna,43 differentially expressed genes(DEGs)were up-regulated,while 79 DEGs were down-regulated.Eleven myosin genes and four troponin I fast skeletal muscle genes(tnni2)were found in DEGs.Among them,four myosin genes were highly expressed in endothermic T.albacores,seven myosin genes and four tnni2 were highly expressed in poikilothermal T.albacores.Eight DEGs were randomly selected for qRT-PCR,and the results were consistent with RNA-seq.The results of GO functional enrichment showed that DEGs were significantly enriched in myosin complex,actin cytoskeleton,motor activity,striated muscle thin filament and others,which were closely related to red muscle motion.And KEGG pathway enrichment suggested that DEGs were enriched in cardiac muscle contraction,pentose phosphate pathway,thermogenesis,etc,which were closely related to muscle motion and energy metabolism.Eleven myosin genes were found differentially expressed during T.albacores development,which meant that the muscle motion of the red muscle might be regulated by myosin genes.Additionally,three tnni2 related with moving were found down-regulated during T.albacores development.There was a large number of vascular network in red muscle of T.albacores.The down-regulating expression of tnni2 may contribute to the establishment of vascular network in red muscle in T.albacores.In this study,the expressions of three genes related to energy metabolism were significantly changed during the"homeothermic"development of T.albacores.They were cytochrome c oxidase subunit 4(cox Ⅳ),ATP-dependent 6-phosphofructokinase(ATP_pfk)and cytochrome c oxidase subunit NDUFA4(Ndufa4).ATP_Pfk and Ndufa4 were downregulated and cox Ⅳ was up-regulated during the"homeothermic"development of T.albacores.ATP_Pfk is a key rate-limiting enzyme in glycolytic pathway.Cox Ⅳ and Ndufa4 are essential for the assembly and function of the cytochrome C oxidases.The endothermic development of T.albacores is a complex process involving multiple tissues and genes.Energy metabolism may play significant roles in"homeothermic"development of T.albacores.The results canl provide important references for further study on the regulation mechanism of development of T.albacores.
万方数据
维普
