| Coilia nasus(junior synonym C.ectenes)is classified under the Clupeiforme,Engraulidae.It is an important commercial anadromous species in the Yangtze River.C.nasus always make anadromous migration for spawning in spring every year and move to sea as juveniles for growth.This fish is called as "Three Delicious Species in the Yangtze River" with Reeve’s shad(Tenualosa reevesii)and obscure pufferfish(Takifugu fasciatus)for its delicacy.However,in recent years,resources of wild C.nasus have been destroyed severely because of over-fishing,severe environmental pollution and over-construction of water conservancy project.Exploration on anadromous mechanism of C.nasus will play active role in recovery of C.nasus resources.At present,there are very few relevant researches of this species,which only focused on olfactory epithelium transcriptome and molecular cloning of relevant genes,e.g.,receptor of olfactory epithelium.Increasing salinity,low temeprature and feed abundance are important environmental factors which juvenile C.nasus will experience during moving to sea.Actually,no research has been available so far about this issue.High-throughput sequencing(the next-generation sequencing)has higher speed,higher accuracy and larger amount of information compared with the first-generation sequencing.It makes researchers to discuss expression of all transcripts at the specific physiological state,and,therefore,is one important method for modern molecular biology.In the present study,we choosed increasing salinity,low temperature and starvation as experimental factors and studied the corresponding effects on the brain and liver transcriptome,so as to discuss the regulation mechanism of juvenile C.nasus under effects of these environmental factors,and to provide theoretical reference for further research on regulation mechanism of C.nasus during its downstream movement.The effect of increasing salinity and low temperature on brain transcriptome of juvenile C.nasus was made.Total RNA was extracted and mRNA was collected,constructed cDNA library,made Illumina sequencing,assembled the transcripts,and made annotation based on NR,SWISSPROT and KOG data bases.Then,It was further made Go enrichment analysis and KEGG pathway analysis,calculated expression level of genes and screened differentially expressed genes and finally made GO and KEGG enrichment analysis on significantly differentially expressed genes.Moreover,4,721 differentially expressed genes were obtained.Among of which,2,028 genes were up-regulated,and 2,693 genes were down-regulated.With further analysis,Top 10 GO annotation terms were obtained.In the subcategory of "Biological process","synaptic transmission" and "neuropeptide signaling pathway" were the most significantly enriched terms;while in the subcategory of"Molecular function","calcium ion binding" and"G-protein coupled receptor activity" were most significantly enriched;as for the subcategory of "cellular component","extracellular region","cell junction","postsynaptic membrane" and "synaptic vesicle membrane" were most obviously enriched.The Top 10 KEGG pathways mainly involved in signal transmission,e.g.,"Neuroactive ligand-receptor interaction","Calcium signaling pathway","Glutamatergic synapse",and immune response e.g.,"Hematopoietic cell lineage".The differentially expressed genes mainly related to the receptors,regulatory proteins and subunits in signal transduction pathway;and the channel protein and transporters in osmoregulation;as well as regulatory factors and enzymes in anti-stress response.Finally,real-time PCR were carried out to make verification,the results were consistent with the transcriptome data,which indicated the accuracy of the transcriptome data in this study.The effect of starvation on liver transcriptome of juvenile C.nasus was conducted based on the aforementioned methods of RNA extraction,cDNA libarary construction,sequencing,de novo assembly,annotation and enrichment analysis on significantly differentially expressed genes.A total of 2,746 differentially expressed genes were obtained.Among of which 1,317 genes were up-regulated and 1,429 genes were down-regulated.With further analysis,the Top 10 GO terms were obtained in the subcategory of "Biological process","Molecular function" and "Cellular component".In "Biological process" category,"DNA integration","immune response","lipid catabolic process","digestion" and"complement activation" were most significantly enriched.In "Molecular function"category,"RNA-directed DNA polymerase activity","aspartic-type endopeptidase activity","nucleic acid binding" and "carbohydrate binding" were the most obviously enriched terms.In the category of "Cellular component",differentially expressed genes mainly involved in"extracellular region","integral component of endoplasmic reticulum membrane","cell surface","receptor complex" were the most significantly enriched terms.Top 10 KEGG pathways mainly involved in four aspects:energy metabolism,e.g.,"fat digestion and absorption","protein digestion and absorption" and "glycolysis/gluconeogenesis";hormone metabolism,e.g.,"steroid hormone biological synthesis";signal transduction,e.g.,"neuroactive ligand-receptor interaction" and "PPAR signaling pathway";immune response,e.g.,"cytokine-cytokine receptor interaction" and "complement and coagulation cascades".Also,the significantly expressed genes were relevant to the above-mentioned categories.In addition,real-time quantitative PCR were performed to make verification.The results were consistent with transcriptome data,indicating the accuracy of transcriptome data of this study.In the investigation on brain transcriptome of the present study,nuclear factor erythroid 2-related factor 2 was screened.It is an anti-stress transcription factor with wide function,and belongs to the most active member of CNC leucine activation factor family.Under the effect of stressor,it may start the transcription of downstream genes to make anti-stress regulatory function.Under effect of increasing salinity and low temperature,molecular cloning,phylogenetic analysis and differential expression were studied on nuclear factor erythroid 2-related factor 2.The results showed that factor had typical conserved motif of basic region leucine zipper and N-glycosylation sites,being the closest phylogenetic relationship with the Atlantic herring Clupea harengus.Meanwhile,it significantly expressed during stress which indicated its positive function in osmoregulation and anti-cold stress.Additionly,aquaporin 1 was also screened in the investigation on brain transcriptome of this study.It is integral membrane protein which assists trans-membrane transportation of water and other small molecules including glycerol and ammonia.It was previously reported that this protein might have important regulatory function during osmoregulation and anti--stress response.Under increasing salinity and low temperature,molecular cloning,phylogenetic analysis and expression were analyzed and the results showed that aquaporin 1 has typical domain of the aquaporin family,e.g.,six trans-membrane regions,highly conserved asparagine-proline-alanine motif.It has the closest phylogenetic relationship with the Charengus,too.It was up-regulated during hyperosmotic and cold stress,suggesting then a possible active role in osmoregulation and anti-stress response.The effect of starvation on liver transcriptome of C.nasus was investigated in the study.The lipoprotein liapse and phosphenolpyruvate carboxykinase were the important regulatory enzymes in glyconeogenesis,and the lipid metabolism pathways was the most significantly enriched.Molecular cloning and expression analysis on them showed that lipoprotein lipase possesses the typical functional domains of lipase family including N-glycolysis sites,binding sites for lipids and triads of aspartic aci-serine-histidine.Phosphoenolpyruvate carboxykinase possesses the conserved domains including specific domain for oxaloacetic binding and two kinase motifs for GTP binding.The enzymes have the closest phylogenetic relationship with those of C.harengus.At starvation of C.nasus,both of the two genes were significantly expressed,implying their positive roles in energy metabolism.In summary,temperature,salinity and starvation were three environmental factors which juvenile Cnasus will experience during catadromous migration,the author for the first time made systematic study on the effect of these factors on juvenile Cnasus based on high-throughput sequencing.At the cooperative stress of salinity and temperature on brain transcriptome,the most signficiantly enriched pathways mainly involved signal transduction and anti-stress.A lot of important differentially expressed genes could be screened.At the stress of starvation on liver transcriptome,the most significantly enriched pathways were relevant to energy metabolism.Furthermore,nuclear factor erythroid 2 related factor 2 and aquaporin 1 were two important regulatory genes screened from brain transcriptome study.This study indicated for the first time that they might play positive regulatory roles in anti-stress response.The transcriptome data have already been submitted to NCBI.Our results will provide valuable genetic information and lay theoretical reference for further study on migration mechanism of C.nasus.Moreover,our results will also provide theoretical reference for further study on migration mechanism of other migratory fish. |
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