بررسی بیان ژن لیزوزیم نوع-C در بافت‌های قدامی کلیه، کبد، پانکراس و طحال مولدین ماهی قره برون (Acipenser persicus Borodin, 1897) با استفاده از روش real time RT-PCR

نوع مقاله: مقاله کامل


1 دانش‌آموخته کارشناسی ارشد شیلات، دانشکده کشاورزی و منابع طبیعی، دانشگاه گنبدکاووس، گلستان، ایران

2 استادیار گروه شیلات، دانشکده کشاورزی و منابع طبیعی، دانشگاه گنبدکاووس، گلستان، ایران

3 گروه بهداشت عمومی و مواد غذایی، دانشکده دامپزشکی، دانشگاه شهید باهنرکرمان، کرمان ایران

4 دانشیار گروه شیلات، دانشکده کشاورزی و منابع طبیعی، دانشگاه گنبدکاووس، گلستان، ایران


لیزوزیم‌ها از مهم‌ترین پروتئین‌های سیستم ایمنی ذاتی در ماهیان به‌عنوان یک پپتید ضد باکتریایی بخصوص در برابر باکتری‌های گرم مثبت شناخته می‌شوند. با توجه به قابلیت انتقال این آنزیم از مولدین به نسل بعد و همچنین کسب پیشرفت‌های اخیر درزمینه ژنتیک مولکولی در استفاده از روش‌های نوین درزمینه آنالیز mRNA با استفاده از روش Real Time PCR جهت اندازه‌گیری میزان بیان ژن‌های مختلف، مطالعه حاضر باهدف ارزیابی میزان بیان ژن لیزوزیم نوع-C در بافت‌های کبد، قدامی کلیه، پانکراس و طحال در ماهیان مولد قره برون (Acipenser persicus) با استفاده از روش مذکور مورد بررسی قرار گرفت. بدین منظور نمونه‌برداری از بافت‌های موردنظر در زمان تکثیر مصنوعی این‌گونه، پس از انجام عملیات تخم‌کشی، اسپرم‌گیری و کشتار از 16 عدد ماهی مولد (هشت عدد ماهی نر، هشت عدد ماهی ماده) انجام شد. RNA کل استخراج و سنتز cDNA از بافت‌های تفکیک‌شده انجام شد. در ادامه بیان mRNA ژن لیزوزیم نوع-C در بافت‌های مختلف با استفاده از تکنیک real time RT- PCR به‌وسیله ژن β-actin به‌عنوان کنترل داخلی مورد ارزیابی قرار گرفت. نتایج به‌دست‌آمده نشان داد ژن لیزوزیم نوع-C در تمام بافت‌های تحت بررسی بیان دارد و حداکثر تعداد نسخه‌های mRNA این ژن در بافت طحال و کمترین تعداد آن در بافت پانکراس مشاهده شد (01/0 > p). درمجموع، با توجه به نقش عوامل مختلف مانند جنسیت، سن، اندازه، فصل، دمای آب، pH، مسمومیت‌ها، عفونت‌ها و درجه عوامل استرس‌زا در میزان فعالیت و یا سطوح آنزیم لیزوزیم که درنهایت ممکن است باعث فعال شدن مستقیم رونویسی ژن لیزوزیم گردند و اهمیت ماهیان مولد خاویاری می‌توان بیان نمود که این ژن به‌خوبی می‌تواند به‌عنوان یک ژن مرجع به‌منظور پیش‌آگاهی، شناسایی و غربالگری ماهیان مولد بیمار و درمان آن‌ها و کنترل محیط‌زیست در این‌گونه مورداستفاده قرار گیرد.



عنوان مقاله [English]

Investigation of the expressions of the C-type Lysozyme gene in head kidney, liver, pancreas and spleen tissues in persian breeding sturgeon (Acipenser persicus Borodin, 1897) by using real time RT- PCR assays

نویسندگان [English]

  • M.R. Bivareh 1
  • H. Gholipour Kannani 2
  • B. Sadeghi 3
  • H. Jafaryan 4
1 Department of Fisheries and Forestry, Faculty of natural resource, Gonbad Kavous University, Golestan, Iran.
2 Assistant professor, Department of Fisheries and Forestry, Faculty of natural resource, Gonbad Kavous University, Golestan,Iran.
3 Food Hygiene and Public Health Department, Faculty of Veterinary Medicine, Shahid Bahonar University of Kerman, Kerman.
4 Associate professor, Department of Fisheries and Forestry, Faculty of natural resource, Gonbad Kavous University, Golestan, Iran.
چکیده [English]

Lysozymes are important proteins of the innate immune system for the defense against bacterial infection, especially gram-positive bacteria in fishes. According to the ability to transfer this gene from breeders to the next generation, as well as recent advantage-ground in molecular genetics on applying novel methods in the field of mRNA analysis using real time RT-PCR to measure the expression of the different genes, the present study was conducted to evaluate the expression of the C- type lysozyme gene in tissues of liver, head of kidney, pancreas and spleen in breeding of Acipenser persicus. For this purpose, tissue sampling was done after artificial breeding from 16 fish (8 males and 8 females). The tissues were transported to the laboratory in liquid nitrogen. Total RNA was isolated and cDNA synthesis was performed from resected tissues. The mRNA expression of the C-type Lysozyme gene in different tissues were detected by real time quantitative PCR with β-actin gene as an internal control. Expression of the c-type lysozyme gene was detected in all tested tissues. The maximum copy numbers of the mRNA expression of the C-type Lysozyme gene were detected in spleen tissue and the lowest expression of this enzyme was determined in the pancreas tissue (p < 0.01). Overall, considering the role of different factors such as sex, age, size, season, water temperature, pH, toxicants, infections and type of stressors in the lysozyme activity or level, which may have eventually caused the direct activation of transcription of lysozyme gene, as well as considering the importance of breeding of sturgeons, we could claim that this gene can well be used as a reference gene for the prognosis and screening of immune deficiency and control of their environment in A. persicus.

کلیدواژه‌ها [English]

  • Gene expression
  • Lysozyme-C
  • real time RT-PCR
  • Acipenser persicus

1- Akbarzadeh, A. 2011. Expression of the gene encoding the enzyme L-gulo in the early stages of growth and differentiation of Persian sturgeon. PHD Thesis. Department of Fisheries. Faculty of Natural Resources. University of Tehran. Iran (in Persian).
2- Bu, X., X. Du, W. Zhou, X. Zhao and J. Wang. 2008. Molecular cloning, recombinant expression and characterization of lysozyme from Chinese shrimp (Fenneropenaeus chinensis). Chinese Journal of Biotechnology 24(5): 723-732.
3- Bustin, S. A. 2000. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. Journal of Molecular Endocrinology 25: 169-193.
4- Cecchini, S., M. Paciolla, E. Biffali, M. Borra, M.V. Ursini and M.B. Lioi. 2013. Ontogenetic profile of innate immune related genes and their tissuespecific expression in brown trout (Salmo trutta (Linnaeus, 1758)). Fish and Shellfish Immunology 35(3): 988-992.
5- Cortopassi, G.A., and N. Arnheim. 1990. Detection of a specific mitochondrial DNA deletion in tissues of older humans. Nucleic Acids Research 18(23): 6927-6933.
6- Dautigny, A., E. M. Prager, D. Pham-Dinh, J. Jollès, F. Pakdel, B. Grinde and P. Jollès. 1991. cDNA and amino acid sequences of rainbow trout (Oncorhynchus mykiss) lysozymes and their implications for the evolution of lysozyme and lactalbumin. Journal of Molecular Evolution 32(2): 187-198.
7- Di Luzio, N.R. 1979. Lysozyme activity: An index of macrophage functional status. In Lysozymes in biology and pathology J.T. Dingle, P. J. Jacques and J.H., Shaw (eds). North Holland Publishing Company. Amsterdam. New York. Oxford: 447-462.
8- Fernandes, J.M.O., G.D. Kemp and V.J. Smith. 2004. Two novel muramidases from skin mucosa of rainbow trout (Oncorhynchusmykiss). Comparative Biochemistry and Physiology -Part B: Biochemistry and Molecular Biology 138(1): 53-64.
9- Fernandez-Trujillo, M.A., J. Porta, M. Manchado, J.J. Borrego, M.C. Alvarez and J. Be jar. 2008. c- Lysozyme from Senegalese sole (Solea senegalensis): cDNA cloning and expression pattern. Fish and Shellfish Immunology 25(5): 697–700.
10- Fu, G.H., Z.Y. Bai, J.H. Xia, F. Liu, P. Liu and G.H. Yue. 2013. Analysis of Two Lysozyme Genes and Antimicrobial Functions of Their Recombinant Proteins in Asian Sea bass. PLoS ONE 8(11): e79743.
11- Gao, F.y., L. Qu, S.G. Yu, X. Ye, Y.Y. Tian, L.L Zhang, J.J. Bai and M. Lu. 2012. Identification and expression analysis of three c-type lysozymes in Oreochromis aureus. Fish and Shellfish immunology 32(5): 779-788.
12- Hew, C.L., G.L. Fletcher and P.L. Davies. 1995. Transgenic salmon: tailoring the genome for food production. Journal of tailoring the genome for food production. Journal of Fish Biology 47(sA): 1-19.
13- Hikima, J., I. Hirono and T. Aoki. 1997. Characterization and expression of C-type lysozyme cDNA from Japanese flounder (Paralichthys olivaceus). Molecular Marine Biology and Biotechnology 6(4): 339–44.
14- Hikima, S., J. Hikima, J. Rojtinnakorn, I. Hirono and T. Aoki. 2003. Characterization and function of kuruma shrimp lysozyme possessing lytic activity against Vibrio species. Gene 316(16): 187-195.
15- Irwin, D.M., and Z. Gong. 2003. Molecular evolution of vertebrate goose-type lysozyme genes. Journal of Molecular Evolution 56(2): 234-242.
16- Jimenez-Cantizano, R.M., C. Infante, B. Martin-Antonio, M. Ponce, I. Hachero, J.I. Navas, and M. Manchado. 2008. Molecular characterization, phylogeny, and expression of c-type and g-type lysozymes in brill Scophthalmus rhombus. Fish and Shellfish Immunology 25(1-2): 57-65.
17- Kaizu, A., F.F. Fagutao, H. Kondo, T. Aoki and I. Hirono. 2011. Functional Analysis of C-type Lysozyme in Penaeid Shrimp. The Journal of Biological Chemistry 286(52): 44344–44349.
18- Lie, O. 1980. Genetic variation in the serum lysozyme activity in cattle. Acta Veterinaria Scandinavica 21: 448-450.
19- Liu, F., and Z. Wen. 2002. Cloning and expression pattern of the lysozyme C gene in zebra fish. Mechanisms of Development 113(1): 69-72.
20- Mikryakov V.R., T.B. Lapirova, M. Soltani, A.S. Mavrin, and G.A. Vinogradov. 2002. Influence of sublethal concentrations of some heavy metal salts (Hg, Cd and Cu) on the contents of lysozyme in tissues of sturgeon fingerlings (Acipenser baeri). Bulletin of the European Association of Fish Pathologists 22(1): 15-20.
21- Mikryakov, V.R., and T.B. Lapirova. 1997. Influence of salts of some heavy metals on picture of white blood cells of Siberian sturgeon fingerling (Acipenser baeri, Brandt). Journal of Problems Ichthyology 37(4): 538-542.
22- Nazari, R.M., M. Modanloo, M.R. Ghomi and M.R. Ovissipor. 2010. Application of synthetic hormone LHRH-A2 on the artificial propagation of Persian sturgeon (Acipenser persicus). Aquaculture international 18 (5): 837–841.
23- Niu, D., E. Peatman, H. Liu, J. Lu, H. Kucuktas, S. Liu, F. Sun, H. Zhang, T. Feng, Z. Zhou, J. Terhune, G. Waldbieser, J. Li and Z. Liu. 2011. Microfibrillar-associated protein 4 (MFAP4) genes in catfish play a novel role in innate immune responses. Developmental and Comparative Immunology 35(5): 568-579.
24- Peatman, E., and Z. Liu. 2007. Evolution of CC chemokines in teleost fish: a case study in gene duplication and implications for immune diversity. Immunogenetics 59(8): 613-623.
25- Rajabzadeh, E., F. Asadi, A. Jolodar, M.R. Seyfi, A. Shapouri, M. Soltani and M. Eslami. 2012. Molecular cloning and expression of g-type Lysozyme from the skin mucus of common Carp (Cyprinus carpio) and its lytic activity. Advanced Studies in Biology 4(9): 419– 435.
26- Rice, C. D. 2001. Fish immunotoxicology: understanding mechanisms of action.pp.96-138. In: D. Schlenk and W. H. Benson (ed.), Target Organ Toxicity in Marine and Freshwater Teleosts, vol. 2. Taylor & Francis, London: CRC Press.
27- Saurabh, S., and P.K. Sahoo. 2008. Lysozyme: an important defence molecule of fish innate immune system; Aquaculture Reserch 29223–239.
28- Serra, C., F. Vizoso, L. Alonso, J.C. Rodríguez, L.O. González, M. Fernández, M.L. Lamelas, L.M. Sánchez, J.L. García-Muñiz, J. Baltasar and A. Medrano. 2002. Expression and prognostic significance of lysozyme in male breast cancer. Breast Cancer Research, 4:16.
29- Soltani, M., and R. Pourgholam. 2007. Lysozyme activity of grass carp (Ctenopharingodon idella) following exposure to sublethal concentrations of organophosphate, diazinon. Journal of Veterinary Research University of Tehran bilingual 62(2): 49-52.
30- Torsteinsdottir, I., L. Hakansson, R. Hallgren, B. Gudbjornsson, N.G. Arvidson and P. Venge. 1999. Serum lysozyme: a potential marker of monocyte/macrophage activity in rheumatoid arthritis. Rheumatol 38: 1249-1254.
31- Wang, Q., Y. Wang, P. Xu and Z. Liu. 2006. NK-lysin of channel catfish: gene triplication, sequence variation, and expression analysis. Molecular Immunology 43(10): 1676-1686.
32- Wen, Y., and D.M. Irwin. 1999. Mosaic evolution of ruminant stomach lysozyme genes. Molecular Phylogenetics and Evolution 13(3): 474-482.
33- Yousif, A., L. Albright and T. Evelyn. 1994. In vitro evidence for the antibacterial role of lysozyme in Salmonid eggs. Diseases of Aquatic Organisms 19: 15–19.
34- Yuan, J.S., A. Reed, F. Chen and C.N. Stewart. 2006. Statistical analysis of real-time PCR data. BMC Bioinformatics 22(7): 85.
35- Zhou, Z., H. Liu, S. Liu, F. Sun, E. Peatman, H. Kucuktas, L. Kaltenboeck, T. Feng, H. Zhang, D. Niu, J. Lu, G. Waldbieser and Z. Liu. 2012. Alternative complement pathway of channel catfish (Ictalurus punctatus): molecular characterization, mapping and expression analysis of factors Bf/C2 and Df. Fish and Shellfish Immunology 32(1): 186-195.