Molecular Cloning, Expression Pattern And In Vitro Recombinant Expression Production Of Orexin From Cynoglossus Semilaevis

In order to investigate the underlying mechanisms for food intake regulation of tongue sole(Cynoglossus semilaevis), the full-length c DNA of the key endocrine factor orexin was isolated using RACE methods and its spatial and temporal expression patterns during embryonic development and early growth stages were investigated by quantitative real-time PCR(q PCR) method. The possible role of orexin in early life stages was discussed by combining q PCR analysis, radio immunoassay and histological observation. Furthermore, in vitro prokaryotic expression system of orexins were constructed and biologically active recombinant proteins were obtained. These results are helpful for better understanding of the role of orexin in the regulation of food intake, growth and reproduction in Cynoglossus semilaevis. The main results are as follows: 1. Structure and ultrastructure of alimentary canal of Cynoglossus semilaevisThe structure and ultrastructure of alimentary canal of Cynoglossus semilaevis was investigated using anatomy, histological section and electronic microscopy methods. The alimentary canal of Cynoglossus semilaevis was divided into five parts including oropharyngeal cavity, esophagus, esophagus and intestinal junction, foregut and hindgut. The number of mucosal folds, the height of mucosal folds, the thickness of muscle layer(longitudinal muscle and circular muscle), the density of mucus cells etc. in each segment of alimentary canal were measured and analyzed. The results showed that there is no obvious morphological stomach in Cynoglossus semilaevis due to lacking of a typical histological structure(a cystic structure named oesogaster), which is formed in posterior part of esophagus post intaking food. The goblet cells initially appeared in front segment of esophagus, type Ⅲ mucous cell is the dominant cell type in the whole esophagus. The mucous cells in oesogaster belong to type Ⅲ mucous cells, whereas type Ⅳ mucous cells are dominant in foregut, and hindgut segment mainly with type Ⅱ mucous cells. The present results would provide basic knowledge for better understanding of digestion mechanism and manufacture of commercial diet for Cynoglossus semilaevis. 2. Molecular cloning and spatio-temporal expression of orexin m RNA in Cynoglossus semilaevisThe full-length c DNA encoding orexin was first isolated from tongue sole Cynoglossus semilaevis hypothalamus using RACE methods. The orexin c DNA is 734 bp in length and encoding a 139 amino acids preprohormone. The predicted orexin A mature peptide is 43 amino acids and orexin B mature peptide is 28 amino acids in length. The tissue distribution results showed that orexin m RNA exhibited highest expression level in brain, and relatively lower expression levels were found in stomach, gill and spleen. orexin m RNA was not detected in unfertilized eggs and early embryos, and it was firstly detected in the embryos at neurula stage, coinciding with the development of the nervous system. However, at post-embryonic stages it increased with growth and peaked at 180 d juvenile stage. These results showed that orexin might play important roles in the early growth and development of Cynoglossus semilaevis. 3. Prokaryotic expression and bioactivity analysis of recombinant orexin from Cynoglossus semilaevisThe mature peptide domains of orexin genes were cloned from Cynoglossus semilaevis. The recombinant plasmids constructed with the matured peptides fragments and the prokaryotic expression vector p ET-32 a were transformed into E. coli BL21(Trans Gen) cells which could be induced by IPTG to produce special fusion polypeptides containing 6×His tags at the N-terminus. The recombinant orexin A and orexin B proteins in form of inclusion bodies proximately account for 52.8% and 43.5% of the whole bacterial protein respectively under the best induce conditions including time, temperature and IPTG concentration, which are confirmed by the SDS-PAGE analysis and the Sigma Scan software. Western blotting analysis indicated fusion protein had the antigenicity to His6 antibody. Supernatant after crushing, purified using Ni2+-NTA affinity chromatography, then purified protein with molecular weight of 24.9 k Da and 21.14 k Da were obtained. Treatment of hypothalamus with recombinant orexin of Cynoglossus semilaevis indicates that orexins can significantly influence on expression of NPY m RNA, orexin m RNA and secretion of NPY peptide in hypothalamus. Therefore, the obtained recombinant orexins have biological activity in the present study. These results would be helpful for better understanding the roles of orexins in feeding regulation and development of high-efficient feeding promotion additives for aquaculture of Cynoglossus semilaevis.