| Turbot(Scophthalmus maximus)is a kind of cold-water fish which originated from the northeastern part of the Atlantic Ocean and the European coast.Turbot was introduced into China by Academician Lei Jilin in 1992,and it has become an important factory-cultivated fish on the north coast in China.However,the high temperature of the northern coastal in China increased morbidity,fecundity and mortality,which is not conducive to factory culture and benefit of turbot.We begin to genetically improve the temperature-resistance traits of turbot to selected high-temperature resistant new lines with excellent growth performance from 2007.At present,the temperature tolerance of the breeding offspring has been significantly improved.In order to speed up the breeding process,we need to excavate the functional genes related to high temperature response and analyze the molecular mechanism of turbot in response to high temperature stress.By analyzing the transcriptome sequencing data of the skin tissue under the high temperature stress conditions,several stress-related stress resistance genes are screened.Then these genes are resolved by gene cloning,tissue expression analysis and prokaryotic expression.We look forward to understanding the molecular mechanisms of heat stress response of turbot in a genome-wide to lay a foundation for a comprehensive understanding of the molecular mechanism of stress resistance of turbot and provide a new theoretical basis and research direction for the development of new markers for high temperature genetic breeding of turbot.1.By analyzing the turbot high-temperature skin transcriptome database to eliminate the low-quality data and mix splicing the clean reads to obtain 217,609 Unigene as the reference sequence for subsequent analysis.We use Nr,Nt,Swiss-Prot,KEGG,GO,KOG and Pfam these seven functional databases to annotate functional transcripts of the spliced genomics,and classifies the functions of Unigene by KEGG,GO and KOG.The DESeq software was used to screen differential genes in the experimental and control groups and 3,497 differential genes were screened,including 2043 up-regulated genes and 1,454 down-regulated genes.According to the gene annotation,10 genes related to high temperature stress are further screened out.After PCR amplification and sequencing,six genes with correct sequence were obtained: PDIA3,PDIA6,FTH1,HSP90?,SERPINH1,and HSP70.2.Using RACE technology to clone the above six genes,the complete gene sequences of PDIA3 and PDIA6 were obtained.The PDIA3 cDNA sequence is 2083 bp in length and 1479 bp in the open reading frame.The 5? non-coding region(UTR)and 3? non-coding region are 85 bp and 519 bp.The ORF region of the gene encodes a 492 amino acid residue.The protein has a relative molecular weight of 54.92 kD,the theoretical isoelectric point of 5.40 and the signal peptide consisting of 17 amino acids.PDIA3 sequence has been submitted to NCBI(Genbank accession number No.MG765516).The full-length cDNA sequence of PDIA6 is 2018 bp,with an open reading frame of 1326 bp,a 5' non-coding region(UTR)and a 3' non-coding region of 95 bp and 597 bp,.The ORF region of PDIA6 encodes a 441 amino acid residue.The protein composition has a relative molecular weight of 47.72 kD,the theoretical isoelectric point of 5.01 and the signal peptide consisting of 19 amino acids.PDIA6 sequence has been submitted to NCBI(Genbank Accession No.MG787152).3.The relative expression levels of six genes in eight tissues such as liver,skin,and palate of turbot,and differential expression changes of six genes after temperature stress at 14°C,20°C,25°C,and 28°C are analyzed by qPCR.The results are as follows: PDIA3 gene has the highest expression level in the liver(P<0.05),followed by the intestinal,and the expression level in the brain is lowest.The expression of PDIA3 gene in liver and intestine increased with increasing temperature and reached the maximum at 28°C(3 times,3.2 times).PDIA6 gene expression was highest in the liver(P<0.05)and lowest in the heart.With the increase of temperature,the expression level in liver and kidney also increased correspondingly,reaching the maximum at 28°C(2.8 times,2.5 times).FTH1 gene has the highest expression in the liver(P<0.05),followed by the spleen and heart and the lowest expression is in the sputum and intestine.With the increase of temperature,the expression of FTH1 in liver and heart increased accordingly.At 28°C,it reaches the maximum value(2.8 times,4.1 times).The expression of HSP90? was highest in the heart and brain(P<0.05),followed by liver,lowest in the kidney.The expression of HSP90? in heart and spleen increased first and then decreased with increasing temperature,reaching the maximum at 25°C.The values are 3.6 and 4.2 times.SERPINH1 gene has the highest expression in the liver(P<0.05),followed by the heart,and the lowest expression is in the corpus callosum.The expression of SERPINH1 in heart and spleen increase significantly with increasing temperature and reached at 28°C.The maximum values are 5.3 and 7.2 times.HSP70 gene expression is highest in the spleen(P<0.05),followed by the gill,lowest in the skin.With the increase of temperature,the expression of HSP70 in sputum and spleen increased first and then decreased,reaching the maximum at 25°C.The maximum value reaches 3.7 and 4.7 times.4.Using the cloned complete PDIA3 gene sequence,primers with restriction sites(BamHI,HindIII)were designed,and the mature peptide coding fragment was amplified by PCR and ligated with the pET-28 a vector after double digestion.The pET-28aPDIA3 expression vector is successfully constructed.The pET-28a-PDIA3 was transformed into DE3 E.coli to induce expression.The induction conditions were 37°C shaking culture to OD600 nm of about 0.6,adding IPTG until the final concentration is 1mmol/L and the culture is continued for 4 h.The expression of the target protein was detected by SDS-PAGE electrophoresis,but it was mainly found in inclusion bodies and was not easily purified.Through optimization experiments,different induction conditions were changed.Finally,it was found that when the temperature was 28° C,the rotation speed was 150 rpm and the IPTG concentration was 0.5 mmol/L,the expression level of the target protein in the supernatant was the largest,which was favorable for the subsequent protein purification and functional verification. |
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