| Turbot(Scophthalmus maximus)belongs to the genus Smilax,Aphididae,and genus,and was introduced into China in the 1990 s.It has become one of the leading varieties of factory farming in the northern coastal areas of China.Turbot is a cold-temperature fish,which requires strict environmental indicators such as temperature,and is suitable for growing water temperature of 12~19 °C;usually under good aeration and running conditions,the high temperature can withstand 25~26 °C,but Poor tolerance to high temperatures in summer.Due to the limitation of water temperature conditions,the northern part of China mainly adopts the model of “cultured greenhouse + deep well seawater” for breeding.However,in recent years,with the shortage of underground seawater and the emergence of the north-south relay breeding mode,new varieties of high temperature resistant to turbot in aquaculture production The demand is increasing.In addition,with the development of molecular technology,the application of molecular biology methods to accelerate the molecular mechanism of the heat-resistant traits of turbot has great research significance for breeding high-temperature resistant cultured species [1,4].In this research paper,a high-throughput sequencing method was established to establish a kidney transcriptome database under the heat stress condition of turbot,in which the differential genes related to the ubiquitin protease system were significantly enriched,and molecular cloning and functional verification of key genes were carried out.The molecular mechanism of heat stress provides data reference and theoretical support.At the same time,based on the high temperature resistant QTL obtained in this laboratory,shared QTL marker mining is carried out to provide a reliable marker basis for molecular marker breeding research.The specific research results are as follows:1 To explore the molecular mechanism of high temperature tolerance of turbot,screen high temperature-related genes,and use the high-throughput sequencing platform(IlluminaHiSeq-2500)to transcribe the transcriptome of five different high-temperature treatment groups of turbot kidneys for biological information.Analysis,including GO(gene function annotation),SSR(simple repeat sequence)analysis.The main results obtained are as follows: The total number of Unigenes obtained by assembly is 68525,the length range is 201-23456 bp,the average length is 1124 bp,and the N50 length is 2316 bp.Unigenes was sequenced and functionally annotated in Nr,Swissprot,KEGG,KOG,and GO databases.A total of 25,498 annotations were made,among which Unigenes were most commented out by Nr database.According to GO function classification,they were divided into cell components and molecular functions.And biological processes 3 categories,including 56 functional groups,a large number of Unigenes related to cell processes,metabolic processes,catalytic activity,biological regulation,stress response.Unigenes was further annotated by pathways,belonging to 218 metabolic pathways,classified into five classes of KEGG pathways: metabolic pathways,genetic information processing,cellular processes,environmental information,and biological systems.A total of 65 transcription factors were detected by transcription factor analysis,and the C2H2 zinc finger protein family had the largest number of genes.By analyzing the results of gene expression profiles under different temperature stresses,there were significant differences among different temperature groups.In the different temperature stress groups,the difference between the 20 °C group and the 28 °C group was the largest,and the differential genes reached 4734,of which up-regulation There are 3386 genes and 1348 genes are down-regulated.This study established the transcriptome database of the heat stress kidney of turbot,which provided a rich data reference for the molecular mechanism of high temperature stress in turbot.2.Recent studies have shown that organisms produce abnormal or damaged proteins under stress conditions,accumulate in the body,affect cell metabolism,and ultimately destroy the integrity of cell structure and function.As the accumulation of abnormalities increases,Protein degradation plays a crucial role in maintaining cell homeostasis;the ubiquitin proteasome system plays an active role in stress perception,signal transduction,and activation of stress-reflecting pathways.In the previous chapter,the high-throughput sequencing platform was used to obtain the transcriptome sequencing data of five different temperature-treated turbot kidneys.Through bioinformatics analysis,a large number of significant differentially expressed genes(DGEs)were obtained,and the KEGG pathway was significantly enriched.The key genes in the ubiquitin proteasome system(ubiquitin-binding enzyme E2 H,ubiquitin ligase E3-CBL)were used for full-length cloning and expression analysis.The full-length cDNA sequence of the UBC2 UBR2 H gene was 745 bp.The gene ORF encodes a protein consisting of 183 amino acid residues with a relative molecular mass of 23.95 kDa and a theoretical isoelectric point of 4.87.The full-length cDNA sequence of the turbot CBL gene is 2263 bp.The gene ORF encodes a protein consisting of 528 amino acid residues with a relative molecular mass of 72.19 kDa and a theoretical isoelectric point of 7.89.In this study,based on the high-density genetic linkage map and high temperature QTL analysis of the previously constructed turbot,15 SNPs related to high temperature tolerance were screened out,and SNP amplification primers for HRM detection were designed.Correct.The HRM small fragment method was used to classify the 15 high temperature-related SNP loci in four families and combined with the previously mapped family related trait data for QTL locus verification.QTL verification results showed that the CC genotype in M19194,the CC genotype in M33439,and the TT and AT genotypes in M12898 were significantly correlated with high temperature traits.The sharing of shared QTL between varieties can expand the use space of QTL markers and reduce the workload and cost of QTL marker positioning for new varieties to reconstruct maps.Provide data assistance for molecularly assisted breeding production. |
PDF Full Text Request