Analisis Gen tufA Secara In Silico Untuk Primer Identifikasi Mikroalga Trebouxiophyceae

Megga Ratnasari Pikoli, Mahsa Nuraini Syahda, Festy Auliyaur Rahmah, Suharti Suharti



Penelitian belakangan ini menunjukkan bahwa di antara mikroalga yang memiliki kandungan lipid tinggi untuk dimanfaatkan sebagai bahan baku biodiesel, termasuk ke dalam kelas Trebouxiophyceae.  Kesederhanaan sel dan bentuknya yang mudah berubah menjadikannya sulit diidentifikasi secara morfologis. Oleh karena itu, identifikasinya perlu didampingi dengan metode molekuler yang mengamplifikasi gen dengan polymerase chain reactions (PCR). Metode PCR membutuhkan primer yang membatasi area pada DNA yang akan diamplikasi. Gen yang berpotensi dijadikan penanda identifikasi adalah tufA karena memiliki urutan yang lestari. Penelitian ini bertujuan mengajukan primer berdasarkan gen tufA untuk identifikasi Trebouxiophyceae. Sekuen gen tufA dikumpulkan dari database, disejajarkan, dan diamati area yang lestari untuk diambil kandidat primer. Kemudian primer forward dan reverse dipasang-pasangkan sambil diperiksa untuk diperoleh kandidat dengan sifat-sifatnya terbaik. Ada 5 pasangan kandidat yang dihasilkan yang kemudian diperiksa spesifisitasnya dalam menjaring anggota genus dari Trebouxiophyceae, dan juga yang bukan Trebouxiophyceae (Chlorophyceae dan Ulvophyceae) sebagai pembanding. Pasangan primer yang terbaik diusulkan dari penelitian ini adalah pasangan primer tufA. Trebo1 yang terdiri atas primer forward 5’-GAAAGTGTTGCTGGTGATAATGTTGG-3’ dan reverse 5’-GGAGTATGTCGACCACCTTCTTC-3’ yang menjaring 75% Trebouxiophyceae di GenBank. Pasangan primer ini menjaring lebih banyak Trebouxiophyceae dibandingkan dengan primer tufA yang pernah dipublikasi, namun memerlukan optimasi kondisi PCR untuk meminimalkan potensi terjadinya struktur sekunder. Dengan demikian, area lestari pada gen tufA berpotensi dijadikan primer untuk identifikasi Trebouxiophyceae.


Recent research showed that microalgae having high lipid content to be used as raw materials for biodiesel belong to the class Trebouxiophyceae.  The simplicity of the cell and its easily changing shape make it difficult to identify morphologically. Therefore, its identification needs to be accompanied by molecular methods that amplify genes with polymerase chain reactions (PCR). The gene that could potentially be used as an identification marker is tufA because it has a conserved sequence. This study aims to propose a primer pair based on the tufA gene for the identification of Trebouxiophyceae. The sequences of the tufA gene were collected from a database of Trebouxiophyceae, aligned, and observed in conserved areas for primer candidates. Then the primary forward and reverse are mounted while checking for the candidate with the best properties. Five candidate pairs were produced, which were then tested for their specificity to bring in members of the Trebouxiophyceae, as well as non-Trebouxiophyceae (Chlorophyceae and Ulvophyceae) as comparisons. The best proposed primary pairs from this study were the primer pair tufA.Trebo1 which consists of the forward 5’-GAAAGTGTTGCTGGTGATAATGTTGG-3’ and the reverse 5‘-GGAGTATGTCGACCACCTTCTTc-3’ that capture 75% of the Trebouxiophyceae in the GenBank. This primer pair contains more Trebouxiophyceae than any previously published tufA primer but requires optimization of PCR conditions to minimize the occurrence of secondary structures. Therefore, the conserved area in the tufA gene has the potential to be used as a primer for identifying Trebouxiophyceae.


Identifikasi; Mikroalga; Primer PCR; Trebouxiophyceae; tufA; Identification; Microalgae; PCR Primer

Full Text:



CBOL Plant Working Group. (2009). A DNA barcode for land plants Communicated by. Proceedings of the National Academy of Sciences, 106(31), 12794-12797.

Crossley, B. M., Bai, J., Glaser, A., Maes, R., Porter, E., Killian, M. L., … Toohey-Kurth, K. (2020). Guidelines for sanger sequencing and molecular assay monitoring. Journal of Veterinary Diagnostic Investigation, 32(6), 767-775. doi: 10.1177/1040638720905833.

Ebertz, A. (2022). Primer design guide – the top 5 factors to consider for optimum performance. Retrieved from

El-Sheekh, M. M., El-Mohsnawy, E., Mabrouk, M. E. M., & Zoheir, W. F. (2020). Enhancement of biodiesel production from the green microalga Micractinium reisseri via optimization of cultivation regimes. Journal of Taibah University for Science, 14(1), 437-444. doi: 10.1080/16583655.2020.1745505.

Famà, P., Wysor, B., Kooistra, W. H., & Zuccarello, G. C. (2002). Molecular phylogeny of the genus Caulerpa (Caulerpales, Chlorophyta) inferred from chloroplast tufA gene. Journal of Phycology, 38, 1040-1050.

Fang, L., Leliaert, F., Zhang, Z. H., Penny, D., & Zhong, B. J. (2017). Evolution of the Chlorophyta: Insights from chloroplast phylogenomic analyses. Journal of Systematics and Evolution, 55(4), 322-332). doi: 10.1111/jse.12248.

Ghosh, A., Khanra, S., Mondal, M., Halder, G., Tiwari, O. N., Saini, S., … Gayen, K. (2016). Progress toward isolation of strains and genetically engineered strains of microalgae for production of biofuel and other value added chemicals: A review. Energy Conversion and Management, 113, 104-118. doi: 10.1016/j.enconman.2016.01.050.

Green, M. R., & Sambrook, J. (2018). Hot start polymerase chain reaction (PCR). Cold Spring Harbor Protocols, 2018(5), 346-349. doi: 10.1101/pdb.prot095125.

Gulbrandsen, S., Andresen, I. J., Krabberød, A. K., Br, J., & Shalchian-Tabrizi, K. (2021). Phylogenomic analysis restructures the Ulvophyceae. Journal of Phycology, 57(4), 1223-1233. doi: 10.1111/jpy.13168-20-145.

Hall, J. D., Fučíková, K., Lo, C., Lewis, L. A., & Karol, K. G. (2010). An assessment of proposed DNA barcodes in freshwater green algae. Cryptogamie Algologie, 31(4), 529-555.

Heesch, S., Pažoutová, M., Moniz, M. B. J., & Rindi, F. (2016). Prasiolales (Trebouxiophyceae, Chlorophyta) of the Svalbard Archipelago: Diversity, biogeography and description of the new genera Prasionella and Prasionema. European Journal of Phycology, 51(2), 171-187. doi: 10.1080/09670262.2015.1115557.

Kazi, M. A., Reddy, C. R. K., & Jha, B. (2013). Molecular phylogeny and barcoding of Caulerpa (Bryopsidales) based on the tufA, rbcL, 18S rDNA and ITS rDNA genes. PLoS ONE, 8(12). doi: 10.1371/journal.pone.0082438.

Kirkendale, L., Saunders, G. W., & Winberg, P. (2013). A molecular survey of Ulva (Chlorophyta) in temperate Australia reveals enhanced levels of cosmopolitanism. Journal of Phycology, 49(1), 69-81. doi: 10.1111/jpy.12016.

Krivina, E., Temraleeva, A., & Sinetova, M. (2022). New species Micractinium kostikovii (Chlorellaceae, Trebouxiophyceae) from Russia. Phycological Research, 70(1), 22-34. doi: 10.1111/pre.12469.

Lawton, R. J., Mata, L., de-Nys, R., & Paul, N. A. (2013). Algal bioremediation of waste waters from land-based aquaculture using Ulva: Selecting target species and strains. PLoS ONE, 8(10). doi: 10.1371/journal.pone.0077344.

Li, S., Sun, H., Hu, Y., Liu, B., Zhu, H., Hu, Z., & Liu, G. (2020). Four new members of foliicolous green algae within the Watanabea clade (Trebouxiophyceae, Chlorophyta) from China. Journal of Eukaryotic Microbiology, 67(3), 369-382. doi: 10.1111/jeu.12787.

Li, S., Tan, H., Liu, B., Zhu, H., Hu, Z., & Liu, G. (2021). Watanabeales ord. nov. and twelve new species of Trebouxiophyceae (Chlorophyta). Journal of Phycology, 57(4), 1167-1186. doi: 10.1111/jpy.13165-20-198.

Liu, B., Liu, X., Hu, Z., Zhu, H., & Liu, G. (2016). Phylogenetic position and morphological observation of the ctenocladus circinnatus borzi, a rare green alga from Changtang Plateau, China. Phytotaxa, 260(1), 75-82. doi: 10.11646/phytotaxa.260.1.8.

Milano, J., Ong, H. C., Masjuki, H. H., Chong, W. T., Lam, M. K., Loh, P. K., & Vellayan, V. (2016). Microalgae biofuels as an alternative to fossil fuel for power generation. Renewable and Sustainable Energy Reviews, 58, 180-197. doi: 10.1016/j.rser.2015.12.150.

Moniz, M. B. J., Guiry, M. D., & Rindi, F. (2014). TufA phylogeny and species boundaries in the green algal order Prasiolales (Trebouxiophyceae, Chlorophyta). Phycologia, 53(4), 396-406. doi: 10.2216/13-233.1.

Okonechnikov, K., Golosova, O., Fursov, M., Varlamov, A., Vaskin, Y., Efremov, I., … Tleukenov, T. (2012). Unipro UGENE: A unified bioinformatics toolkit. Bioinformatics, 28(8), 1166-1167. doi: 10.1093/bioinformatics/bts091.

Oliveira, C. Y. B., D’Alessandro, E. B., Filho, N. R. A., Lopes, R. G., & Derner, R. B. (2021). Synergistic effect of growth conditions and organic carbon sources for improving biomass production and biodiesel quality by the microalga Choricystis minor var. minor. Science of the Total Environment, 759. doi: 10.1016/j.scitotenv.2020.143476.

Pikoli, M. R., Sari, A. F., Solihat, N. A., & Permana, A. H. (2019). Characteristics of tropical freshwater microalgae Micractinium conductrix, Monoraphidium sp. and Choricystis parasitica, and their potency as biodiesel feedstock. Heliyon, 5(12). doi: 10.1016/j.heliyon.2019.e02922.

Pröschold, T., & Darienko, T. (2020). Choricystis and Lewiniosphaera gen. nov. (Trebouxiophyceae Chlorophyta), two different green algal endosymbionts in freshwater sponges. Symbiosis, 82(3), 175-188. doi: 10.1007/s13199-020-00711-x.

Qiao, K., Takano, T., & Liu, S. (2015). Discovery of two novel highly tolerant NaHCO3 Trebouxiophytes: Identification and characterization of microalgae from extreme saline-alkali soil. Algal Research, 9, 245-253. doi: 10.1016/j.algal.2015.03.023.

Sáez, A. G., Zaldivar-Riverón, A., & Medlin, L. K. 2008. Molecular systematics of the Pleurochrysidaceae, a family of coastal coccolithophores (Haptophyta). Journal of Plankton Research, 30(5), 559-566.

Saunders, G. W., & Kucera, H. (2010). An evaluation of rbcL, tufA,UPA, LSU and ITS as DNA barcode markers for the marine green macroalgae. Cryptogamie, Algologie, 31(4), 487-528.

Saunders, G. W., & McDevit, D. C. (2012). Methods for DNA barcoding photosynthetic Protists emphasizing the macroalgae and Diatoms. In W. J. Kress, & D. L. Erickson (Eds.), DNA barcodes-methods in molecular biology (pp. 207-222). Totowa, NJ: Humana Press (Springer Science).

Sayers, E. W., Cavanaugh, M., Clark, K., Ostell, J., Pruitt, K. D., & Karsch-Mizrachi, I. (2019). GenBank. Nucleic Acids Research, 47(D1), D94-D99. doi: 10.1093/nar/gky989.

Shaikh, K. M., Nesamma, A. A., Abdin, M. Z., & Jutur, P. P. (2019). Molecular profiling of an oleaginous trebouxiophycean alga Parachlorella kessleri subjected to nutrient deprivation for enhanced biofuel production. Biotechnology for Biofuels, 12(1). doi: 10.1186/s13068-019-1521-9.

Sharma, S., Sutar, R. R., Parida, A., & Bast, F. (2020). DNA barcoding and ITS-tufA multi-local molecular phylogeny of nitrophilic alga Prasiola crispa growing on penguin guano at Larsemann Hills, Eastern Antarctica. Czech Polar Reports, 11(2), 194-202. doi: 10.5817/CPR2021-2-13.

Shuba, E. S., & Kifle, D. (2018). Microalgae to biofuels: ‘Promising’ alternative and renewable energy, review. Renewable and Sustainable Energy Reviews, 81(May 2017), 743-755. doi: 10.1016/j.rser.2017.08.042.

Tale, M., Ghosh, S., Kapadnis, B., & Kale, S. (2014). Isolation and characterization of microalgae for biodiesel production from Nisargruna biogas plant effluent. Bioresource Technology, 169, 328-335. doi: 10.1016/j.biortech.2014.06.017.

Tartar, A., & Boucias, D. G. (2004). The non-photosynthetic, pathogenic green alga Helicosporidium sp. has retained a modified, functional plastid genome. FEMS Microbiology Letters, 233(1), 153-157. doi: 10.1016/j.femsle.2004.02.006.

Utomo, D. H., Ichsan, M., & Putri, J. F. (2019). Prinsip dasar desain primer dengan bioinformatika: Vol cetakan pertama. Global Science.

Vieira, H. H., Bagatini, I. L., Guinart, C. M., & Vieira, A. A. H. (2016). tufA gene as molecular marker for freshwater Chlorophyceae. Algae, 31(2), 155-165. doi: 10.4490/algae.2016.31.4.14.

Wynne, M. J., Verbruggen, H., & Angel, D. L. (2009). The recognition of Caulerpa integerrima (Zanardini) comb, et stat. nov. (Bryopsidales, Chlorophyta) from the Red Sea. Phycologia, 48(4), 291-301. doi: 10.2216/08-78.1.

Yang, Y., Li, M., Li, H., Li, X. Y., Lin, J. G., Denecke, M., & Gu, J. D. (2020). Specific and effective detection of anammox bacteria using PCR primers targeting the 16S rRNA gene and functional genes. Science of the Total Environment, 734. doi: 10.1016/j.scitotenv.2020.139387.

Ye, J., Coulouris, G., Zaretskaya, I., Cutcutache, I., Rozen, S., & Madden, T. L. (2012). Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction. Retrieved from

Yee, W. (2016). Microalgae from the Selenastraceae as emerging candidates for biodiesel production: A mini review. World Journal of Microbiology and Biotechnology, 32(4), 1-11. doi: 10.1007/s11274-016-2023-6.

DOI: Abstract - 0 PDF - 0


  • There are currently no refbacks.

This work is licensed under a CC-BY- SA.

Indexed By:

/public/site/images/rachma/logo_moraref_75 /public/site/images/rachma/logo_google_scholar_75_01 /public/site/images/rachma/logo_isjd_120 /public/site/images/rachma/logo_garuda_75 /public/site/images/rachma/logo_crossref_120/public/site/images/rachma/logo_base_2_120 /public/site/images/rachma/neliti-blue_75   /public/site/images/rachma/dimensions-logo_120