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
Abstract：Edwardsiella 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.
[1] 李雪, 陈松林, 杨长庚, 等. 2016. 半滑舌鳎转化生长因子TGF-β1和TGF-β3基因的克隆及受哈维氏弧菌感染后表达分析[J]. 农业生物技术学报, 24(6): 889-898. (Li X, Chen S L, Yang C G, et al.2016. Molecular cloning and expression analysis of transforming growth factor TGF-β1 and TGF-β3 in half-smooth tongue sole (Cynoglossus semilaevis) following stimulation with Vibrio harveyi. Journal of Agricultural Biotechnology, 24(6): 889-898.) [2] 王杉, 徐文腾, 李明, 等.2020. 斑石鲷TGF-β1基因克隆和表达分析[J].渔业科学进展,41(3): 78-87. (WANG Shan, XU Wenteng, LI Ming, et al.Molecular cloning and expression pattern analysis of tgf-β1 in spotted knifejaw (Oplegnathus punctatus)[J]. Progress in Fishery Sciences, 2020, 41(3): 78-87.) [3] Arcamone N, Lucini C, Borzacchiello G, et al.2005. Distribution of NGF and NT-3-like protein immunoreactivity in the teleost kidney[J]. Microscopy Research and Technique. 66(1): 17-24. [4] Bang J, Chun S, Park S, et al.1992. Studies on the biochemical and serological characteristics of Edwardsiella tarda isolated from cultured flounder, Paralichthys olivaceus[J]. Journal of Fish Pathology. 5(1): 29-35. [5] Bradshaw R A.1983. Neurobiology: What cloned genes can tell us about nerve growth factor[J], Nature, 303(5920): 751. [6] El-Hashim A Z, Jaffal S M.2017. Cough reflex hypersensitivity: A role for neurotrophins[J]. Experimental Lung Research, 43(2): 93-108. [7] Fariñas I.1999. Neurotrophin action during the development of the peripheral nervous system[J]. Microscopy Research and Technique. 45(4-5): 233-242. [8] Girolamo PD, Arcamone N, Lucini C, et al.2000. The teleost kidney expresses Trk neurotrophin receptor-like proteins[ J]. Anatomy and Embryology, 201(5): 429-433. [9] Gunn-Moore F J, Williams A G,TAVARÉ Jeremy M.1997. Analysis of mitogen-activated protein kinase activation by naturally occurring splice variants of TrkC, the receptor for neurotrophin-3[J]. Biochemical Journal, 322(1): 193-198. [10] Hallböök F, Ibáñez C F, Persson H, et al.1991. Evolutionary studies of the nerve growth factor family reveal a novel member abundantly expressed in Xenopus ovary[J]. Neuron, 6(5): 845-858. [11] Hempstead B L, Martin-Zanca D, Kaplan D R, et al.1989. High-affinity NGF binding requires coexpression of the trk proto-oncogene and the low-affinity NGF receptor[J], Nature, 350(6320): 678-683. [12] Huang E J, Reichardt L F.2001. Neurotrophins: Roles in neuronal development and function[J]. Annual Review of Neuroence. 24(1): 677-736. [13] Isshiki T, Nishizawa T, Kobayashi T, et al.2001. An outbreak of VHSV (Viral hemorrhagic septicemia virus) infection in farmed Japanese flounder Paralichthys olivaceus in Japan[J]. Diseases of Aquatic Organisms. 47(2): 87-99. [14] Jeanneteau F, Deinhardt K, Miyoshi G, et al.2010. The MAP kinase phosphatase MKP-1 regulates BDNF-induced axon branching[J]. Nature Neuroscience. 13(11):1373-1379. [15] Ji Y M, Hong Y K, Kong H J, et al.2014. A cDNA microarray analysis to identify genes involved in the acute-phase response pathway of theolive flounder after infection with Edwardsiella tarda[J]. Veterinary Immunology and Immunopathology, 161(1-2): 49-56. [16] Klein R, Parada L F, Coulier F, et al.1989. TrkB, a novel tyrosine protein kinase receptor expressed during mouse neural development[J]. Embo Journal, 8(12): 3701-3709. [17] Maisonpierre P C, Belluscio L, Squinto S, et al.1990. Neurotrophin-3: A neurotrophic factor related to NGF and BDNF[J]. Science, 247(4949): 1446-1451. [18] Margarita Behrens M, Uta Strasser, Doug Lobner, et al.1999. Neurotrophin mediated potentiation of neuronal injury[J]. Microscopy Research and Technique. 45(4-5): 276-284. [19] Mizuno Y, Takada H, Urakami K, et al.2000. Neurotrophin-3 levels in cerebrospinal fluid from children with bacterial meningitis, viral meningitis, or encephalitis[J]. Journal of Child Neurology. 15(1): 19-21. [20] Moustafa E M M, Naota M, Takehito Morita, et al. 2010. Pathological study on the scuticociliatosis affecting farmed Japanese flounder (Paralichthys olivaceus) in Japan[J]. The Journal of veterinary medical science. 2010,72(10):1359-1362. [21] Reichardt L F.2006. Neurotrophin-regulated signalling pathways[J]. Philosophical Transactions of the Royal Society of London. Series B, Biological sciences. 361(1473): 1545-1564. [22] Sariola H.2001. Neurotrophic factors in non-neuronal tissues[J]. Cellular & Molecular Life ences Cmls. 58(8): 1061-1066. [23] Seikai T.2002. Flounder culture and its challenges in Asia.[J]. Reviews in Fisheries Science. 10(3-4): 421-432. [24] Shao C W, Bao, B L, Xie Z Y, et al.2017. The genome and transcriptome of Japanese flounder provide insights into flatfish asymmetry[J]. Nature Genetics, 49(1): 119-124. [25] Sofroniew M W, Howe C L, Mobley W C.2001. Nerve growth factor signaling, neuroprotection and neural repair[J]. Annual Review of Neuroence. 24(1): 1217-1281. [26] Tanja Maehr, Maria M. Costa, Jose L. González Vecino, et al.2013. Transforming growth factor-β1b: A second TGF-β1 paralogue in the rainbow trout (Oncorhynchus mykiss) that has a lower constitutive expression but is more responsive to immune stimulation[J]. Fish and Shellfish Immunology, 34(2): 420-432. [27] Tessarollo L.1998. Pleiotropic functions of neurotrophins in development[J]. Cytokine and Growth Factor Reviews. 9(2): 125-137. [28] Vega J A, García-Suárez O, Hannestad J, et al.2003. Neurotrophins and the immune system[J]. Journal of anatomy, 203(1): 1-19. [29] Zapata A, Diez B, Cejalvo T, et al.2006. Ontogeny of the immune system of fish[J]. Fish and Shellfish Immunology,20(2): 126-136. [30] Zhou X F, Rush R A.1994. Localization of neurotrophin-3-like immunoreactivity in peripheral tissues of the rat[J]. Brain Research. 621(2): 189-199.
