| Osmotic adjustment of fish is an important dynamic equilibrium process,which is extremely important for the survival of fish.But so far,the research progress on fish salinity adaptation mechanism is still in its infancy.Trachidermus fasciatus is a migratory fish in rivers and seas,with a wide range of salt suitable.In this study,T.fasciatus is selected as the research object to carry out research on the adaptation mechanism of fish salinity.This paper is the first to carry out the whole genome sequencing of T.fasciatus,using bioinformatics technology to assemble and annotate it,evaluate the genome quality,analyze the genome structure,protein-coding gene composition and its characteristics,and compare T.fasciatus from the perspective of comparative genome The T.fasciatus genome has undergone significant expansion and contraction gene family categories and positively selected genes were identified and functionally analyzed,and genes related to salinity tolerance were screened to lay the foundation for further research on the genomics mechanism of salinity adaptation;Transcriptomics analysis of T.fasciatu tissues under salinity stress,obtained rich transcriptome information of T.fasciatus,screened and identified candidate genes that have undergone expansion and contraction or are being positively selected,and expressed differentially which related to salinity adaptation.The key genes of salinity tolerance were further analyzed,the main research results are as follows:1.Determine the genomic sequence of T.fasciatus by de novo sequencing technology,and assemble the genomic sequence by Wtdbg2 software to obtain the complete genomic sequence of T.fasciatus.The genome size of T.fasciatus obtained by assembly is 556 Mb,and the length of Contig N50 evaluation is more than 10 Mb.The BUSCO2.0 evaluation results show that the completeness of T.fasciatus genome is 94.4% in the eukaryotic gene set and 97.3% in the metazoan gene set.The quality of the assembled genome is high.Annotation of repeated sequences in T.fasciatus genome by RepeatMasker4.0.5 showed that: de novo de novo prediction and prediction of protein level and nucleotide level,a total of 125 M repeated sequences were annotated,accounting for 22.54% of the entire genome size,Its proportion is similar to that of other fishes(25.2% Trilobus,19.76% Flower Bass,19.03% Small Yellow Croaker,etc.).Using a combination of homology alignment and de novo de novo prediction,a total of 19,499 protein-coding genes were annotated in the T.fasciatus genome,and these genes were functionally annotated.The results showed that97.7% of the genes can be used in InterPro,TrEMBL,GO,KEGG,Swissprot The COG six functional gene libraries were successfully annotated,and the remaining 441 genes thatcould not be annotated may be unique to T.fasciatus.The genomic information of T.fasciatus obtained in this study laid a good foundation for the next step of screening candidate genes for salinity tolerance by comparative genomics research.The software OrthoMCL1.4 was used to cluster the gene families of T.fasciatus and Xu's scorpionfish,yellowtail yellowtail,large yellow croaker,scorpionfish,red spotted grouper and zebrafish,and use RAxML8.0.8 for single copy genes The phylogenetic tree was constructed by the alignment sequence of the family,and the results showed that the relationship between T.fasciatus fish and Xu's Pingmo was the closest.Using the codeml tool in PAML4 to detect the genes that were positively selected in T.fasciatus,405 candidate genes were finally identified,and based on the results of previous studies,we screened 30 genes related to osmotic pressure regulation,including Hsp70(Heat shock protein 70),OSTF1(Osmotic stress transcription factor 1),IGF-1(Insulin-like growth factor),IGFBP-1(Insulin-like growth factor-binding protein 1),V-ATPase(Vacuolar-type H+-ATPase),Claudin-4,etc.,these genes may have an important regulatory role for T.fasciatus in adapting to changes in environmental osmotic pressure.2.Through high-throughput transcriptome sequencing technology,analyze the transcriptome of T.fasciatus brain,gill,intestine,and kidney under salinity stress,and compare with the control group to screen and identify the differentially expressed expressions after salinity stress.gene.The results showed that compared with the control group,brain,gill,intestine,kidney and other tissues showed 7915(upregulated3155/downregulated 4760),2517(upregulated 758/downregulated 1759),2394(upregulated 1304/downregulated 1090),1707(Up-regulated 618/down-regulated 1089)differentially expressed genes.The comprehensive analysis of GO function and KEGG pathway enrichment shows that the significantly differentially expressed genes in brain tissue are mainly enriched in signal transmission pathways such as hormone secretion,synaptic transmission,and ion pathway,and related genes such as GH(Grouth hormone),CA The expression of carbonic anhydrase(Carbonic Anhydrase,),SGK1(Serum/glucocorticoid-regulated kinase 1)and other genes were significantly up-regulated;the significantly differentially expressed genes in intestinal tissue were mainly enriched in the pathways of protein,peptide,ammonium metabolism,etc.On the top,the expression of related genes such as IGFBP-I was significantly up-regulated and FABP(Fatty acid-binding protein 6)was significantly down-regulated;the significantly differentially expressed genes in gill tissue were mainly enriched in glucose metabolism,immune response,phagocytosis,In apoptosis and other pathways,the expression of related genessuch as Na+/K+ATP(NKA),CRP(C-reactive protein)and other genes has been significantly up-regulated;the significantly differentially expressed genes in kidney tissue are mainly enriched in cytokines Interactions,immune responses,and receptor binding pathways,the expression of genes related to them,such as PRLR(Prolactin receptor),GADD45?(growth arrest and DNA damage inducible protein 45?),were significantly up-regulated.This result suggests that the brain,Gill,intestine,kidney and other four tissues play an important role in the salinity stress of T.fasciatus.Compare the results of genes related to expansion or contraction and positively selected osmotic pressure regulation obtained with the previous selection with differentially expressed genes to obtain 23 salinity-related genes that are both significantly changed and differentially expressed in stress experiments,including V-ATPase,Claudin-4,Hsp70,IGFBP-I,AQP1 and other common genes have been used as biomarkers in many studies to explore the response mechanism of fish to changes in environmental osmotic pressure.It is speculated that these genes play a role in the evolution process of T.fasciatus salinity adaptation Key genes.3.For the first time in this paper,the complete genome assembly annotation of T.fasciatus and the transcriptome sequencing have been completed.The salt-tolerance genes of T.fasciatus have been screened and their salt-tolerance regulation mechanism has been analyzed.This will be useful for T.fasciatus.Genomics,transcriptomics and molecular biology research provide corresponding data support,and at the same time lay a solid theoretical foundation for the future selection of T.fasciatus and other fish wide-salt strains. |
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