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
Abstract:Transforming growth factor β (TGF-β) superfamily plays critical roles in immune cell functional regulation, cell proliferation and differentiation, tissue wound repair. The half-smooth tongue sole (Cynoglossus semilaevis), with excellent properties such as fast growth, individual large, meat delicious and nutrient-rich, is an important economic marine fish species in the North of China. It has become a new kind of excellent breeding object in recent years. But with the enlargement of the farming scale, a variety of infectious diseases is becoming more and more frequently, especially the bacterial disease such as the Vibrio harveyi infection, fast onset and spread quickly. These diseases bring great economic losses to the aquaculture industry. Therefore, in order to carry out the half-smooth tongue sole immune genetic research and explore its immune response mechanism with Vibrio harveyi infection, the TGF-β1 and TGF-β3 genes were cloned from liver in the half -smooth tongue sole. Sequence analysis showed that TGF-β1 and TGF-β3 amino acid sequence consisted of multiple N-glycosylation sites and a TGF-β family signature. Analysis of phylogenetic tree indicated that TGF-β1 and TGF-β3 of the half-smooth tongue sole had a high homology with that of fish. The qRT-PCR analysis showed that TGF-β1 and TGF-β3 expressed in a diverse array of tissues including heart, kidney, intestine, muscle, skin, brain, gill, liver and spleen of healthy half-smooth tongue sole and there were significant differences. The highest levels of TGF-β1 were detected in skin, the higher levels were discovered in kidney and the lowest levels were found in muscle. TGF-β3 expressed the highest in skin, higher in brain and the lowest in muscle. After the Vibrio harveyi infection, the expression levels of TGF-β1 showed uptrend in liver, peaked at 48 h after infection, with 3.17 times of the control group, then fell to control levels. In the spleen, TGF-β1 and TGF-β3 showed similar expression trend , rising in the first stage and then decreasing. The expression of TGF-β1 and TGF-β3 reached maximum after infection 24 and 12 h, respectively, with 1.70 times and 1.61 times of the control group (P<0.05). In the kidney, the expression trend of TGF-β1 and TGF-β3 were still similar, both peaked at 12 h after infection, respectively, with 1.23 times and 1.42 times of the control group (P<0.05), then showed a downward trend. In the gill, TGF-β1 expression quantity did not change significantly comparing with the control group. TGF-β3 expression quantity rose in the first stage and then decreased, peaked at 24 h after infection, with 4.71 times of the control group (P<0.05). The above results suggested that TGF-β1 and TGF-β3 may play an important role in the immune against bacterial infections in the half-smooth tongue sole. This study provides a strong evidence for the participation of TGF-β1 and TGF-β3 in the immune regulation mechanism and also a theoretical basis for molecular immune research in the half- smooth tongue sole.
陈松林,田永胜,徐田军,等. 2008. 牙鲆抗病群体和家系的建立及其生长和抗病性能初步测定[J]. 水产学报,32(5):665-673. (Chen S L, Tian Y S, Xu T J, et al. Development and characterization for growth rate and disease resistance of disease-resistance population and family in Japanese flounder (Paralichthys olivaceus)[J]. Journal of Fisheries of China, 32(5): 665-673.)李晖,李健,孙铭,等. 2013. 氟苯尼考对3种海洋致病性弧菌的体外抗菌后效应[J]. 渔业科学进展,34(6):107-111. (Li H, Li J, Sun M, et al. Postantibiotic effects of florfenicol in vitro against three marine pathogenic Vibrios[J]. Progress in Fishery Sciences, 34(6):107-111.)宋江南,李校堃,苏志坚,等. 2006. TGF-β3生物学特性、功能与临床应用前景[J]. 药物生物技术,13(2):154-158. (Song J N, Li X K, Su Z J, et al. TGF-β3 biological characteristics, function and clinical application prospect[J]. Pharmaceutical Biotechnology, 13(2): 154-158. )万瑞景,姜言伟,庄志猛. 2004. 半滑舌鳎早期形态及发育特征[J]. 动物学报,50(1):91-102. (WAN R J, JIANG Y W, ZHUANG Z M. Morphological and developmental characters at the early stages of the tonguefish Cynoglossus semilaevis [J]. Acta Zoologica Sinica, 50(1): 91-102.)王印庚,陈君,潘传燕,等. 2013. 鲆鲽类循环水养殖系统中病原菌的分布及杀除工艺[J]. 渔业科学进展,34(3):75-81. (Wang Y G, Chen J, Pan C Y, et al. Pathogenic bacteria distribution and extermination in the recirculating aquaculture system of flatfish[J]. Progress in Fishery Sciences, 34(3):75-81.)张晓君,秦国民,阎斌伦,等. 2009. 半滑舌鳎病原鳗利斯顿氏菌表型及分子特征研究[J]. 海洋学报,31(5):112-122. (HANG X J, QIN G M, YAN B L, et al. Phenotypic and molecular characterization of pathogenic Listonella anguillarum isolated from half-smooth tongue sole Cynoglossus semilaevis [J]. Acta Oceanologica Sinica, 31(5): 112-122. )Ellis A E. 2001. Innate host defense mechanisms of fish against viruses and bacteria[J]. Developmental & Comparative Immunology, 25(8): 827-839.Garanger E, Boturyn D, Dumy P. 2007. Tumor targeting with RGD peptide ligands-design of new molecular conjugates for imaging and therapy of cancers[J]. Anticancer Agents Med Chem, 7(5): 552-558.Garry D D, Christopher J S. 1999. Genomic organisation of rainbow trout, Oncorhynchus mykiss TGF-β[J]. Developmental & Comparative Immunology, 23(2): 139-147.Haddada G, Haningtona P C, Wilson E C, et al. 2008. Molecular and functional characterization of goldfish (Carassius auratus L.) transforming growth factor beta[J]. Developmental & Comparative Immunology, 32(6): 654-663.Harms C A, Stoskopf K S, Horne W A, et al. 2000. Cloning and sequencing hybrid striped bass (Morone saxatilis×M.chrysops) transforming growth factor-β(TGF-β), and development of a reverse transcription quantitative competitive polymerase chain reaction (RT-qcPCR) assay to measure TGF-β mRNA of teleost fish[J]. Fish & Shellfish Immunology, 10(1): 61-85.Helenius A, Aebi M. 2001. Intracellular function of N-linked glycans[J]. Science, 291(5512): 2364-2369.Kapral M, Strzalka B, Kowalczyk M, et al. 2008. Transforming growth factor β isoforms (TGF-β1, TGF-β2, TGF-β3) messenger RNA expression in laryngeal cancer[J]. American Journal of Otolaryngology, 29(4): 233-237.Kohli G, Hu S, Clelland E. 2003. Cloning of transforming growth factor-beta 1 (TGF-beta 1) and its type II receptor from zebrafish ovary and role of TGF-beta 1 in oocyte maturation[J]. Endocrinology, 144: 1931-1941.Kondaiah P, Taira M, Vempati U D, et al. 2000. Transforming growth factor-β5 expression during early development of Xenopus laevis[J]. Mechanisms of Development, 95(1): 207-209.Kunzmann S, Mantel P Y, Wohlfahrt J G. 2003. Histamine enhances TGF-β mediated suppression of Th2 responses[J]. The FASEB Journal, 17(1): 89-95.Laing K J, Pilstrom L, Cunningham C, et al. 1999. TGF-β3 exists in bony fish[J]. Veterinary Immunology and Immunopathology, 72:45-53.Lee U E, Friedman S L. 2011. Mechanisms of hepatic fibrogenesis[J]. Best Practice & Research Clinical Gastroenterology, 25(2): 195-206.Matejuk A, Dwyer J, Hopke C, et al. 2004. Opposing roles for TGF-β1 and TGF-β3 isoforms in experimental autoimmune encephalomyelitis[J]. Cytokine, 25: 45-51.Medina A, Mancera J M, Manzanares E M, et al. 2015. Identification of Vibrio harveyi proteins involved in the specific immune response of Senegalese sole (Solea senegalensis, Kaup)[J]. Fish & Shellfish Immunology, 47(1): 377-380.Novoa B, Bowman T V, Zon L, et al. 2009. LPS response and tolerance in the zebrafish (Danio rerio)[J]. Fish & Shellfish Immunology, 26(2): 326-331.Paolo B, Paolo G, Guillermo H, et al. 2014. The role of transforming growth factor (TGF)-βin modulating the immune response and fibrogenesis in the gut[J]. Cytokine & Growth Factor Reviews, 25: 45-55.Pelton R W, Dickinson M E, Moses H L,et al. 1990. In situ hybridization analysis of TGF β3 RNA expression during mouse development: Comparative studies with TGFβ1 and 2[J]. Development, 110(2): 609-620.Philip N, Howles M. 1997. Transforming Growth Factor β3: Pharmacological properties and physiological functions[J]. Clinical Immunology Newsletter, 17(8): 109, 114-117.Qian S, Thai NL, Lu L, et al. 1997. Liver transplant tolerance:mechanistic insights from animal models, with particular reference to the mouse[J]. Transplant Reviews, 11: 151-164.Rebbert M L, Bhatia D N, Dawid I B. 1990. The sequence of TGF-β2 from Xenopus laevis. Nucleic Acids Research, 18: 2185-2190.Rudd P M, Elliott T, Cresswell P, et al. 2001. Glycosylation and the immune system[J]. Science, 291(5512): 2370-2376.Sly L M, Rauh M J, Kalesnikoff J, et al. 2004. LPS-induced upregulation of ship is essential for endotoxin tolerance[J]. Immunity, 21(2): 227-239.Tafalla C, Aranguren R, Secombes C J,et al. 2003. Molecular characterisation of sea bream (Sparus aurata) transforming growth factor β1[J]. Fish & Shellfish Immunology, 14:405-421.Wan Y Y, Flavell R A. 2007. T Cell-produced transforming growth factor-β1 controls T cell tolerance and regulates Th1- and Th17-Cell differentiation[J]. Immunity, 26(5): 579-591.Yang M, Zhou H. 2008. Grass carp transforming growth factor-beta 1 (TGF-beta1): molecular cloning, tissue distribution and immunobiological activity in teleost peripheral blood lymphocytes[J]. Molecular Immunology, 45(6): 1792–1798.Zhan X L, Ma T Y, Wu J Y, et al. 2015. Cloning and primary immunological study of TGF-β1 and its receptors TβR I/TβR II in tilapia (Oreochromis niloticus)[J]. Developmental and Comparative Immunology, 51: 134-140.