ISOLASI, IDENTIFIKASI, DAN ANALISIS EKSPRESI GEN PENGKODE GROWTH HORMONE PADA IKAN SIDAT (Anguilla bicolor)
Abstract
Gen hormon pertumbuhan (growth hormone, GH) perlu dipelajari untuk mendukung domestikasi ikan sidat Anguilla bicolor di Indonesia. Penelitian ini bertujuan untuk mengisolasi dan mengidentifikasi gen pengkode GH serta menganalisis ekspresinya pada sampel ikan sidat A. bicolor. Primer untuk amplifikasi gen GH ikan sidat A. bicolor didesain berdasarkan sekuen gen GH dari beberapa spesies ikan sidat lain yang tersedia di GenBank. Sampel cDNA hipofisa dan otak ikan sidat digunakan untuk amplifikasi gen GH. Hasil amplifikasi disekuensing dan hasilnya dianalisis menggunakan analisis BLAST, multiple sequences alignment, hormon signature, dan filogenetik. Analisis ekspresi gen GH dilakukan menggunakan teknik realtime PCR dengan metode delta-delta CT pada 14 sampel ikan sidat (23,1 ± 19,6 g; 24,5 ± 4,2 cm). Fragmen DNA sepanjang 486 bp berhasil diamplifikasi dan disekuensing. Sekuen gen GH A. bicolor memiliki similaritas nukleotida sebesar 98,49-99,14% jika dibandingkan dengan gen GH ikan sidat A. anguilla, A. australis, dan A. japonica. Sekuen parsial tersebut secara in silico diketahui mengkode bagian dari protein GH sepanjang 155 asam amino (aa). Sekuen asam amino protein GH A. bicolor sangat mirip dengan sekuen spesies ikan sidat lain dengan hanya tiga perbedaan asam amino dan membentuk satu percabangan pada pohon filogenetik. Tingkat ekspresi gen GH pada sampel ikan sidat memiliki variasi yang tinggi. Hasil penelitian ini dapat menjadi dasar untuk studi selanjutnya khususnya berkaitan dengan peran gen GH dalam pertumbuhan ikan sidat A. bicolor.
The growth hormone (GH) gene needs to be studied to support the domestication of the Anguilla bicolor eel in Indonesia. This study aimed to isolate and identify the GH coding gene and analyze its expression in samples of the eel A. bicolor. Primers for amplification of the GH gene of A. bicolor eels were designed based on GH gene sequences from several other eel species available in GenBank. Pituitary and brain cDNA samples of eel were used for GH gene amplification. The amplification results were sequenced and the results were analyzed using BLAST analysis, multiple sequence alignment, hormone signature, and phylogenetic analysis. The GH gene expression analysis was carried out using the real-time PCR technique with the delta-delta CT method on 14 eel samples (23.1 ± 19.6 g; 24.5 ± 4.2 cm). The 486 bp DNA fragment was successfully amplified and sequenced. The GH gene sequence of A. bicolor has a nucleotide similarity of 98.49-99.14% when compared with the GH gene of other eel species A. anguilla, A. australis, and A. japonica. This partial sequence was found in silico to code 155 amino acids (aa) GH protein. The amino acid sequence of the A. bicolor GH protein is very similar to that of other eel species with only three amino acid differences and forms one branch on the phylogenetic tree. The expression level of the GH gene in eel samples had high variations. The results of this study could be a basis for further studies, especially regarding the role of the GH gene in the growth of the eel A. bicolor.
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Affandi, R., Budiardi, T., Wahju, R. I., & Taurusman, A. A. (2013). Pemeliharaan ikan sidat dengan sistem air bersirkulasi. Jurnal Ilmu Pertanian Indonesia, 18(1), 55–60.
Ahlina, H. F., Sudrajat, A. O., Budiardi, T., & Affandi, R. (2015). Induksi pematangan gonad secara hormonal pada ikan sidat, Anguilla bicolor bicolor McClelland 1844 dengan penggunaan pregnant mare serum gonadotropin, anti dopamin, dan recombinant growth hormone. Jurnal Iktiologi Indonesia, 15(3), 209–221. https://doi.org/10.32491/jii.v15i3.57
Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D. J. (1990). Basic local alignment search tool. Journal of Molecular Biology, 215(3), 403–410. https://doi.org/10.1016/S0022-2836(05)80360-2
Arief, M., Pertiwi, D., & Cahyoko, Y. (2011). Pengaruh pemberian pakan buatan, pakan alami, dan kombinasinya terhadap pertumbuhan, rasio konversi pakan dan tingkat kelulushidupan ikan sidat (Anguilla bicolor). Jurnal Ilmiah Perikanan dan Kelautan, 3(1), 61–66.
Blasco, J., Vélez, E. J., Perelló-Amorós, M., Azizi, S., Capilla, E., Fernández-Borràs, J., & Gutiérrez, J. (2021). Recombinant bovine growth hormone-induced metabolic remodelling enhances growth of gilthead Sea-bream (Sparus aurata): insights from stable isotopes composition and proteomics. International Journal of Molecular Sciences, 22(23). https://doi.org/10.3390/ijms222313107
Brooks, C. L. (2012). Molecular mechanisms of prolactin and its receptor. Endocrine Reviews, 33(4), 504–525. https://doi.org/10.1210/er.2011-1040
Daza, D. O., & Larhammar, D. (2018). Evolution of the growth hormone, prolactin, prolactin 2 and somatolactin family. General and Comparative Endocrinology, 264, 94–112. https://doi.org/https://doi.org/10.1016/j.ygcen.2018.01.007
Degani, G., Tzchori, I., Yom-Din, S., Goldberg, D., & Jackson, K. (2003). Growth differences and growth hormone expression in male and female European eels [Anguilla anguilla (L.)]. General and Comparative Endocrinology, 134(1), 88–93. https://doi.org/https://doi.org/10.1016/S0016-6480(03)00238-7
Dehkhoda, F., Lee, C. M. M., Medina, J., & Brooks, A. J. (2018). The growth hormone receptor: mechanism of receptor activation, cell signaling, and physiological aspects. Frontiers in Endocrinology, 9, 35. https://doi.org/10.3389/fendo.2018.00035
Deng, S.-P., Wu, B., Zhu, C.-H., & Li, G.-L. (2014). Molecular cloning and dimorphic expression of growth hormone (gh) in female and male spotted scat Scatophagus argus. Fisheries Science, 80(4), 715–723. https://doi.org/10.1007/s12562-014-0763-5
Dereeper, A., Guignon, V., Blanc, G., Audic, S., Buffet, S., Chevenet, F., Dufayard, J.-F., Guindon, S., Lefort, V., Lescot, M., Claverie, J.-M., & Gascuel, O. (2008). Phylogeny.fr: robust phylogenetic analysis for the non-specialist. Nucleic Acids Research, 36(2), W465-469. https://doi.org/10.1093/nar/gkn180
Fahmi, M. R. (2015). Konservasi genetik ikan sidat tropis (Anguilla spp.) di perairan Indonesia. Jurnal Penelitian Perikanan Indonesia, 21(1), 45–54. https://doi.org/http://dx.doi.org/10.15578/jppi.21.1.2015.45-54
Fahmi, M. R., & Hirnawati, R. (2010). Keragaman ikan sidat tropis (Anguilla sp.) di perairan Sungai Cimandiri, Pelabuhan Ratu, Sukabumi. Prosiding Forum Inovasi Teknologi Akuakultur, 2010, 1–10.
Faizal, I., Aliah, R. S., Amarullah, M. H., Megawati, N., Sutanti, S., & Alimuddin, A. (2012). Produksi protein rekombinan hormon pertumbuhan ikan kerapu. Jurnal Riset Akuakultur, 7(2), 231–235. http://dx.doi.org/10.15578/jra.7.2.2012.231-235
Forlenza, M., Kaiser, T., Savelkoul, H. F. J., & Wiegertjes, G. F. (2012). The use of real-time quantitative PCR for the analysis of cytokine mRNA levels. In M. De Ley (Ed.), Cytokine Protocols. Methods in Molecular Biology, volume 820 (pp. 7–23). Humana Press. https://doi.org/10.1007/978-1-61779-439-1
Gasteiger, E., Gattiker, A., Hoogland, C., Ivanyi, I., Appel, R. D., & Bairoch, A. (2003). ExPASy: The proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Research, 31(13), 3784–3788.
Hall, T. A. (1999). BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series, 41, 95–98.
Hulo, N., Bairoch, A., Bulliard, V., Cerutti, L., De Castro, E., Langendijk-Genevaux, P. S., Pagni, M., & Sigrist, C. J. A. (2006). The PROSITE database. Nucleic Acids Research, 34(1), D227–D230. https://doi.org/10.1093/nar/gkj063
Jönsson, E., & Bjönsson, B. (2002). Physiological functions of growth hormone in fish with special reference to its influence on behaviour. Fisheries Science, 68(1), 742–748. https://doi.org/10.2331/fishsci.68.sup1_742
Lima, E. R., Freire, R. P., Suzuki, M. F., Oliveira, J. E., Yosidaki, V. L., Peroni, C. N., Sevilhano, T., Zorzeto, M., Torati, L. S., Soares, C. R., Lima, I. D., Kronenberger, T., Maltarollo, V. G., & Bartolini, P. (2023). Isolation and characterization of the Arapaima gigas growth hormone (ag-GH) cDNA and three-dimensional modeling of this hormone in comparison with the human hormone (hGH). Biomolecules, 13(1). https://doi.org/10.3390/biom13010158
Marlina, E., & Handayani, M. (2022). Efektifitas rekayasa media budidaya terhadap respon pertumbuhan pada ikan sidat (Anguila bicolor). Jurnal Riset Rumpun Ilmu Hewani, 1(2), 66–75.
Muttray, A. F., Donaldson, E. M., & Devlin, R. H. (2019). Effects of recombinant salmon type II growth hormone and bovine growth hormone on growth of coho salmon (Oncorhynchus kisutch). Fish Physiology and Biochemistry, 45(3), 1083–1090. https://doi.org/10.1007/s10695-019-00620-x
Ndandala, C. B., Dai, M., Mustapha, U. F., Li, X., Liu, J., Huang, H., Li, G., & Chen, H. (2022). Current research and future perspectives of GH and IGFs family genes in somatic growth and reproduction of teleost fish. Aquaculture Reports, 26, 101289. https://doi.org/https://doi.org/10.1016/j.aqrep.2022.101289
Nguyen, A. T., Damsteegt, E. L., Chia, J. H. Z., Kazeto, Y., & Lokman, P. M. (2022). Effects of gonadotropins, 11-ketotestosterone, and insulin-like growth factor-1 on target gene expression and growth of previtellogenic oocytes from shortfinned eels, Anguilla australis, in vitro. Fish Physiology and Biochemistry, 48(4), 853–867. https://doi.org/10.1007/s10695-022-01090-4
Nugrayani, D., Ekasanti, A., Syakuri, H., Listiowati, E., Soedibya, P. H. T., Marnani, S., Santoso, M., Wijaya, R., Sukardi, P., & Kasprijo, K. (2023). Performa pertumbuhan dan ekspresi gen hormon pertumbuhan sidat (Anguilla bicolor bicolor) stadia elver yang diberi pakan mengandung kromium pikolinat. Jurnal Akuakultur Sungai dan Danau, 8(1), 13–20.
Ozaki, Y., Fukada, H., Tanaka, H., Kagawa, H., Ohta, H., Adachi, S., Hara, A., & Yamauchi, K. (2006). Expression of growth hormone family and growth hormone receptor during early development in the Japanese eel (Anguilla japonica). Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 145(1), 27–34. https://doi.org/https://doi.org/10.1016/j.cbpb.2006.05.009
Politis, S. N., Mazurais, D., Servili, A., Zambonino-Infante, J.-L., Miest, J. J., Sorensen, S. R., Tomkiewicz, J., & Butts, I. A. E. (2017). Temperature effects on gene expression and morphological development of European eel, Anguilla anguilla larvae. PloS One, 12(8), e0182726. https://doi.org/10.1371/journal.pone.0182726
Pratama, I., Prayitno, S. B., & Syakuri, H. (2019). Identification and pevalence of parasites in eel (Anguilla bicolor) captured along migration pathway at Serayu River, Central Java. Omni-Akuatika, 15(1), 81–92.
Rachmawati, F. N., Susilo, U., & Muslih. (2017). Karakteristik reproduksi ikan sidat Anguilla bicolor McClelland , 1844 yang diinduksi GNRH-analog. Jurnal Iktiologi Indonesia, 17(2), 155–163.
Rotwein, P. (2020). Regulation of gene expression by growth hormone. Molecular and Cellular Endocrinology, 507, 110788. https://doi.org/https://doi.org/10.1016/j.mce.2020.110788
Sekar, M., Singh, S. D., & Gupta, S. (2014). Cloning and characterization of Pangasianodon hypophthalmus growth hormone gene and its heterologous expression. Applied Biochemistry and Biotechnology, 173, 1446–1468. https://doi.org/10.1007/s12010-014-0946-7
Setijanto, S., Sulistyo, I., & Eko, B. (2014). Penentuan waktu pengambilan benih dan diet ikan sidat (Anguilla bicolor Mc Clelland) di Sungai Serayu. Universitas Jenderal Soedirman.
Sievers, F., & Higgins, D. G. (2014). Clustal omega. Current Protocols in Bioinformatics, 48, 3-13. https://doi.org/10.1002/0471250953.bi0313s48
Sudo, R., Kawakami, Y., Nomura, K., Tanaka, H., & Kazeto, Y. (2022). Production of recombinant Japanese eel (Anguilla japonica) growth hormones and their effects on early-stage larvae. General and Comparative Endocrinology, 317, 113977. https://doi.org/https://doi.org/10.1016/j.ygcen.2022.113977
Teletchea, F. (2021). Fish domestication in aquaculture: 10 unanswered questions. Animal Frontiers, 11(3), 87–91. https://doi.org/10.1093/af/vfab012
Teletchea, F., & Fontaine, P. (2014). Levels of domestication in fish: Implications for the sustainable future of aquaculture. Fish and Fisheries, 15, 181–195. https://doi.org/10.1111/faf
Triantaphyllopoulos, K. A., Cartas, D., & Miliou, H. (2020). Factors influencing GH and IGF-I gene expression on growth in teleost fish: how can aquaculture industry benefit? Reviews in Aquaculture, 12(3), 1637–1662. https://doi.org/https://doi.org/10.1111/raq.12402
Utomo, D., Alimuddin, A., Sudrajat, A., & Faizal, I. (2011). Produksi dan uji bioaktivitas protein rekombinan hormon pertumbuhan ikan mas. Jurnal Akuakultur Indonesia, 10(1), 44–50.
Yamaguchi, K., Yasuda, A., Kishida, M., Hirano, T., Sano, H., & Kawauchi, H. (1987). Primary structure of eel (Anguilla japonica) growth hormone. General and Comparative Endocrinology, 66(3), 447–453. https://doi.org/https://doi.org/10.1016/0016-6480(87)90256-5
Yeku, O., & Frohman, M. A. (2011). Rapid amplification of cDNA ends (RACE). In H. Nielsen (Ed.), RNA. Methods in molecular biology, volume 703 (pp. 107–122). Humana Press. https://doi.org/10.1007/978-1-59745-248-9_8
Yolla, A., Linggi, Y., & Dahoklory, N. (2020). Pengaruh perbedaan substrat terhadap pertumbuhan ikan sidat (Anguilla bicolor bicolor) di dalam wadah budidaya. Jurnal Aquatik, 3(1), 51–58.
Yuan, X., Lin, Y., Qin, J., Zhang, Y., Yang, G., Cai, R., Liao, Z., Sun, C., & Li, W. (2020). Molecular identification, tissue distribution and in vitro functional analysis of growth hormone and its receptors in red-spotted grouper (Epinephelus akaara). Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 250, 110488. https://doi.org/https://doi.org/10.1016/j.cbpb.2020.110488
DOI: http://dx.doi.org/10.15578/jra.19.1.2024.69-83
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