Analisis Gen tufA Secara In Silico Untuk Primer Identifikasi Mikroalga Trebouxiophyceae
Abstract
Abstrak
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.
Abstract
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.
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DOI: https://doi.org/10.15408/kauniyah.v17i1.35458 Abstract - 0 PDF - 0
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