Gene Cloning, Specific Expression In Different Tissues And Recombinant Protein Preparation Of4Biomarkers In Scophthalmus Maximus

Scophthalmus maximus originated in the eastern Atlantic coastal area. After thetwo decades hard work since turbot was initially introduced into China in1992byacademician Lei Ji-lin, this fish has became the most important economic marineaquaculture breed in the north coastal area of China. Currently, the annual productionof factory farming industry of Scophthalmus maximus is more than60000tons, andthe annual total value is over4billion.This research was mainly focused on screening out functional genes, which wererelated to important physiological regulation in the aquaculture environment, such asfeeding, growth and stress, from the gene expression libraries of various tissues fromScophthalmus maximus which were cultured under the stresses of high temperature,hunger and salinity by our previous work. Furthermore, Biomarkers for quantitativelyevaluating culture physiological indexes of Scophthalmus maximus were selected. Inthis paper, cDNA full-length sequences of4biomarkers were cloned, and theexpression patterns and function were studied, which would build foundation ofmolecular methods for the detection of Scophthalmus maximus culture physiologicalstatus, and to provide the basis for the construction of standardized cultivation modeof Scophthalmus maximus.The main contents of this paper are as follows:1. Full length cDNAs Cloning and sequence analysis of4biomarkers: Orexin,Somatolactin, Calreticulin and GDH in Scophthalmus maximus.Full cDNA sequences of Orexin, Somatolactin, Calreticulin and ORF sequence ofGDH were cloned by RT-PCR and RACE. The full-length of Orexin cDNA was737bp, including an ORF of453bp encoding for150amino acid residues, including47for signal peptide,43for Orexin-A,28for Orexin-B and16for polypeptide of unknown function, and the5’ UTR was15bp, the3’ UTR was269bp. The cDNA ofSomatolactin was1780bp, including a5’ UTR of28bp, a3’ UTR of1050bp and anORF of702bp encoding for233amino acid residues. The full-length of CalreticulincDNA was2158bp, including an ORF of1263bp, which can encode420amino acidresidues, and the5’UTR was59bp,3’UTR was836bp. The ORF ofGDH was1629bpencoding for542amino acid residues.2. Expression analysis of4biomarkers: Orexin, Somatolactin, Calreticulin andGDH in Scophthalmus maximus.By use of Real time PCR technology, we study the expression pattern of Orexin、Somatolactin、Calreticulin and GDH gene in14tissues from turbot. The result showedthat expression of Orexin was very lowly detected in spleen, kidney and not detectedin intestines, in other13tissues, the highest expression was detected in hypothalamus.Expression of Somatolactin was very lowly detected in forebrain, midbrain and notdetected in muscle, in other13tissues, the highest expression was detected inpituitary gland. Expression of Calreticulin was highly detected in4tissues:intestines, liver, head kidney and pituitary gland, but hardly detected in muscle,kidney, forebrain and midbrain. Expression of GDH was highly detected in13varioustissues besides kidney, especially the highest was detected in head kidney.3. Construction of gene engineering vector and preparation of recombinant proteinfor Orexin, Somatolactin and Calreticulin from Scophthalmus maximus.After Orexin、Somatolactin、Calreticulin and GDH gene were respectively insertedinto prokaryotic expression vector PET-24and transformed into E.coli BL21(hostbacterial), the heterologous expression of recombinant protein for Orexin,Somatolactin and Calreticulin from Scophthalmus maximus was performed. The3various fused proteins with His-tag were expressed after inducing with IPTG andpurified with affinity chromatography column. The purified proteins were determinedby SDA-PAGE and the result showed that the expressed protein band of Orexin、Somatolactin and Calreticulin respectively was at16.9kDa,26.9kDa and48.8kDawith predicted size was obtained.This paper mainly studied on the screened biomarkers (Orexin, Somatolactin, Calreticulin and GDH), which were involved in culture physiological regulation suchas feeding, growth and stress. Based on cloning and expression pattern analysis of the4biomarkers, full length of cDNA sequences, express specificity in different tissueswere deeply analyzed, and the recombinant proteins were produced. The biomarkersacquired from this research can not only detect whether Scophthalmus maximus mightact well under the circumstances with environmental stress, but also can be helpful toconstruct the suitable culture environment and standardized cultivation mode forScophthalmus maximus through systematically study the interaction mechanismbetween these biomarkers and culture environment.