Cloning and Expression Analysis of NTF3 Gene in Flounder (Paralichthys olivaceus)
CHEN Zhi-Peng1,2, ZHU Ying2, WANG Lei2, HU Guo-Bin3, CHEN Song-Lin2,*
1 Fisheries College, Ocean University of China, Qingdao 266071, China; 2 Yellow Sea Fisheries Research Institute, Qingdao National Laboratory of Marine Science and Technology Pilot Laboratory of Marine Fishery Science and Food Production Process Functional Laboratory, Chinese academy of fishery sciences, Qingdao 266071, China; 3 College of Marine Life, Ocean University of China, Qingdao 266071, China
AbstractEdwardsiella tarda is the main pathogen causing large-scale death of flounder (Paralichthys olivaceus) in factory farming. Its infectivity is extremely strong. The mortality rate of infected flounder with E. tarda is very high, which seriously impedes the development of flounder industry. In order to study the function of neurotrophin 3 (NTF3) in the innate immunity of P. olivaceus, the full-length cDNA sequence of P. olivaceus NTF3 gene was cloned, with the total length 1 411 bp, in which the 5' noncoding region is 89 bp and the 3' non-coding region is 428 bp.The open reading frame (ORF) of this gene was 894 bp.Which encoded 297 amino acids. The predicted molecular weight (Mw) was about 33.73 ku, and the theoretical isoelectric point (pI) was 9.56. The homology comparison found that the NTF3 gene had a high similarity with different species, and the highest homology was 90.91% with the NTF3 gene of amberjack (Seriola dumerili). The NTF3 gene was expressed in lots of healthy tissues of flounder (blood, liver, spleen, kidney, intestine, skin, gill, heart and brain). The highest expression level was tested in skin followed by blood. To further study the relative expression of NTF3 during E. tarda infection, a bacterial infection experiment was conducted on healthy flounder, and then the relative expression of NTF3 in 4 different tissues (intestine, kidney, spleen and liver) and different time points were compared. It was found that, in the intestine, spleen and liver, the expression levels of NTF3 gene were up-regulated after bacterial stimulation, but in the kidney, the expression levels showed a downward trend. The above results indicated that the NTF3 gene may be involved in the immune process of P. olivaceus against the E.tarda infection, and the relative expression of NTF3 gene is different in different tissues infected by the bacteria, which provides basic research data for further study of the role of NTF3 gene in the immune response process of flounder.
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