KARAKTERISASI FRAGMEN 0.45 kb GEN PENGKODE STILBEN SINTASE (STS) DARI TANAMAN MELINJO (Gnetum gnemon L.)

ELLY RUSTANTI, Dr. Tri Joko Raharjo, M.Si.

2011 | Tesis | S2 Ilmu Kimia

Telah dilakukan penelitian mengenai karakterisasi fragmen 0,45 kb gen pengkode stilben sintase (STS) dari tanaman melinjo (Gnetum gnemon L.). Penelitian ini bertujuan untuk mendapatkan urutan fragmen gen sts tanaman melinjo. Amplifikasi fragmen gen dilakukan dengan RT-PCR (Reversed Transcription Polymerase Chain Reaction) yang dimulai dengan mendesain primer berdasarkan urutan conserve asam amino 10 protein stilben sintase (STS) yang terpublikasi di genebank. Hasil desain primer disintesis dan digunakan untuk amplifikasi RT-PCR dua tahap terhadap total RNA yang diisolasi dari daun melinjo. Fragmen hasil RT-PCR kemudian diisolasi dan dimurnikan untuk dilakukan sekuensing. Hasil sekuensing dianalisis dengan membandingkan urutan nukleotida gen sts di genebank. Hasil desain primer adalah GGF1 (5’GCAACCGTCCTGGCAATCGC 3’) dan GGR1 (5’GTTCCACCTGCGAAGCAGCC 3’) yang diprediksi terdapat dalam posisi 49-69 dan 487-506. Amplifikasi fragmen gen sts menghasilkan beberapa fragmen dengan salah satu fragmen berukuran 0,45 kb sesuai dengan perkiraan ukuran berdasarkan posisi urutan primer gen sts yang lain. Hasil sekuensing fragmen RT-PCR (fragmen 0,45 kb) menunjukkan kemiripan sebesar 77% dengan urutan DNA gen sts Arachis hypogaea L00952. Homologi ini lebih tinggi dibandingkan kemiripan urutan antara gen sts yang biasanya berkisar 66%. Dengan demikian fragmen 0,45 kb hasil amplifikasi benar merupakan bagian gen sts melinjo.

Characterization of 0.45 kb gene fragment encoding stilbene synthase (STS) from melinjo (Gnetum gnemon L.) has been carried out. Study was aimed to obtain nucleotide sequence of melinjo sts gene fragment. A RT-PCR (Reversed Transcriptase Polymerase Chain Reaction) amplification was performed using primers that were designed based on the amino acid conserve sequence of 10 STS protein obtained from the genebank. Two-steps RT-PCR amplification was conducted to a total RNA product isolated from the melinjo leaf. An obtained DNA fragment was purified and sequenced subsequently. The nucleotide sequence was then analyzed and compared to sts Arachis hypogaea to determine its homology to an existed sts gene. The designed primers were GGF1 (5’GCAACCGTCCTGGCAATCGC3’) and GGR1 (5’ GTTCCACCTGCGAAGCAGCC 3’) which were predicted in position 49-69 and 487-506 of sts gene respectively. The RT-PCR results in severals bands with one of them has been isolated as 0.45 kb fragment. The sequence analysis of the 0.45 kb fragment showed that the DNA sequence had 77% similarity to Arachis hypogaea sts gene. This homology was higher than the 66% similarity commonly obtained among sts genes of the various plants. Therefore the amplified fragment could be concluded as part of gene sts melinjo.

Kata Kunci : Gen stilben sintase (sts), RT-PCR, Melinjo (Gnetum gnemon L.)