Analisis Sekuens DNA Penyandi 18S rRNA pada Tumbuhan Cocor Bebek (Kalanchoe x laetivirens)
DOI:
https://doi.org/10.35799/jbl.11.2.2021.32297Abstract
(Article History: Received January 16, 2021; Revised April 6, 2021; Accepted April 13, 2021)
Â
ABSTRAK
Studi mengenai stress pada tumbuhan semakin banyak didasarkan pada ekspresi gen. Gen penyandi 18S rRNA merupakan salah satu anggota dari gen housekeeping yang umum digunakan sebagai kontrol internal. Penelitian ini bertujuan untuk menganalisis sekuens DNA penyandi 18S rRNA pada tumbuhan cocor bebek (K. x laetivirens). Metode penelitian meliputi ekstraksi DNA total dari daun segar menggunakan Mini Kit Genomic DNA Mini Kit Plant (Geneaid). Data sekuen DNA diolah menggunakan program BioEdit, BLASTn dan MEGA 6. Sekuen DNA parsial dari gen penyandi 18S rRNA K. x laetivirens telah diperoleh dengan ukuran 419pb. Sekuen tersebut memiliki kemiripan sebesar 99,28% dengan K. daigremontiana. Kalanchoe x laetivirens membentuk satu kelompok dengan sesama anggota dari famili Crassulaceae dan terpisah dari famili lainnya yang diteliti. Primer 18S rRNA spesifik terhadap cocor bebek telah dirancang, yaitu forward 5’- CAA ATT ACC CAA TCC TGA CA -3’ dan reverse 5’- CCA ACG TAA ATA GGA TCG AA -3’. Sekuen yang diperoleh pada penelitian ini berpotensi sebagai gen referensi setelah dilakukan validasi.
Kata kunci: 18S rRNA; gen housekeeping; Kalanchoe x laetivirens; kontrol internal; PCR
Â
ABSTRACT
Plant stress studies are based on gene expressions. This study aims to analyze the DNA sequence of 18S rRNA in cocor bebek (K. x laetivirens). Methods being out are total DNA isolation using Mini Kit Genomic DNA Mini Kit Plant (Geneaid). The DNA sequence was analyzed utilizing BioEdit, BLASTn and Mega 6 programs. Partial DNA sequence of 18S rRNA in K. x laetivirens has been obtained with 419 bp length. The DNA sequence has 99.28% similarity to K. daigremontiana. Kalanchoe x laetivirens formed one group with another species from the same family, Crassulaceae, based on the DNA sequence of 18S rRNA. A primer pair specific to K. x laetivirens for amplifying 18S rRNA has been designed such as forward 5’- CAA ATT ACC CAA TCC TGA CA -3’ and reverse 5’- CCA ACG TAA ATA GGA TCG AA -3’. This DNA sequence is potentially being employed as an internal control once the validation process completed.
Keywords: 18S rRNA; housekeeping gene; internal control; Kalanchoe x laetivirens; PCRReferences
Ai NS (2012) Evolusi Fotosintesis Pada Tumbuhan. J Ilmiah Sains 12: 28–34.
Chang E, Zhao Y, Wei Q, Shi S, Jiang Z (2016) Isolation of high-quality RNA from Platycladus orientalis and other Cupressaceae plants. Electronic Journal of Biotechnology 23: 21-27.
Chen C, Xie T, Ye S, Jensen AB, Eilenberg J (2015) Selection of reference genes for expression analysis in the entomophthoralen fungus Pandora neoaphidis. Brazilian Journal of Microbiology 47: 259-265.
Christiningrum OD, Budiharjo A, Kusdiyantini E (2016) Karakterisasi Molekuler Tanaman Sambung Nyawa (Gynura Procumbens [Lour.] Merr) Berdasarkan 18S rRNA. Jurnal Biologi 5: 60-70.
Elsima A, Ferniah RS, Kusumaningrum HP (2019) Ekspresi Gen Penyandi Peroksidase Cabai Merah (Capsicum annuum L.) (Caper) sebagai Respons terhadap Fusarium Oxysporum. Jurnal Akademika Biologi 8: 30-35.
Gantasala NP, Papolu PK, Thakur PK, Kamaraju D, Sreevathsa R, Rao U (2013) Selection and Validation of Reference Genes for Quantitative Gene Expression Studies by Real-Time PCR in Eggplant (Solanum melongena L). BioMed Central 6: 1-11.
Kazmi I, Khan R, Singh R, Chauhan M, Anwar F, Bist T (2012) Brophyllum Pinnatum A Review. Int. J. of Research in Biological Sciences 2: 143-149.
Kuchipudi SV, Tellabati M, Nelli RK, White GA, Perez BB, Sebastian S, Slomka MJ, Brookes SM, Brown IH, Dunham SP, Chang KC (2012) 18S rRNA is a Reliable Normalisation Gene for Real Time PCR Based on Influenza Virus Infected Cells. BioMed Central 9: 1-7.
Kwon O, Ogino K, Ishikawa H (1991) The longest 18s ribosomal RNA Ever Known Nucleotide Sequence and Presumed Secondary Structure of the 18s rRNA of the Pea Aphid, Acyrthosiphon pisum. J. Biochem 202 : 827-833.
Latief A (2012) Obat Tradisional. EGC, Jakarta.
Meyer A,Todt C, Mikkelsen NT, Lieb B (2010) Fast evolving 18S rRNA sequences from Solenogastres (Mollusca) Resist Standard PCR Amplification and Give New Insights Into Mollusk Substitution Rate Heterogeneity. BioMed Central 10: 2-12.
Roslim DI (2017). Identification of Pandan Plant (Benstonea sp) from Riau, Indonesia Using Three DNA Barcodes. SABRAO J Breed. and Genet. 49(4): 346-360.
Shaw JMH (2008) An investigation of the cultivated Kalanchoe daigremontiana group, with a checklist of Kalanchoe cultivars. Hanburyana 3: 17–79.
