Preparasi Talus dan Sumber Eksplan Steril Kappaphycus alvarezii

5 PEMELIHARAAN LANJUT EKSPLAN Kappaphycus alvarezii TRANSGENIK DAN ANALISIS EKSPRESI mRNA ABSTRAK Adanya ekspresi mRNA gen yang ditransfer menunjukkan potensi tinggi diperoleh karakter yang diinginkan pada organisme transgenik. Penelitian tahap ini dilakukan pemeliharaan lanjut Kappaphycus alvarezii transgenik dan kontrol, serta menganalisis tingkat ekspresi mRNA gen κ-Carrageenase. Pemeliharaan lanjut eksplan transgenik dilakukan secara semi-steril pada wadah pembesaran botol kerucut volume 1500 ml dengan aerasi. Analisis ekspresi mRNA dari dua tunas transgenik dan satu tunas non-transformasi kontrol. Ekstraksi RNA total dilakukan menggunakan TRIzol ® Reagent, dan sintesis DNA komplementer cDNA dilakukan menggunakan Iscript TM cDNA Synthesis Kit dengan teknik reverse transcriptase PCR RT-PCR. Kuantifikasi tingkat ekspresi gen κ-Car dilakukan dengan quantitative real time PCR qrt-PCR. Hasil pemeliharaan lanjut menunjukkan sintasan eksplan K. alvarezii transgenik sebesar 100, eksplan kandidat transgenik lainnya sebesar 36, dan kontrol sebesar 43, dengan jumlah tunas 2-4 per eksplan dan panjang tunas 5-18 mm. Analisis ekspresi K. alvarezii transgenik menghasilkan tingkat ekspresi gen κ- Carrageenase setelah dinormalisasi terhadap ekspresi gen Actin adalah masing- masing sebesar 0,983 untuk transgenik T1 dan sebesar 0,962 untuk transgenik T2. Dengan demikian, gen κ-Carrageenase diekspresikan dengan level relatif sama pada K. alvarezii transgenik Kata kunci: analisis ekspresi gen, cycle of threshold, qrt-PCR REARING OF TRANSGENIC Kappaphycus alvarezii EXPLANT AND GENE EXPRESSION ANALYSIS ABSTRACT Presence mRNA expression of transferred gene indicates a high potency to achieve a characteristic target on transgenic organism. This research was conducted to analyze the level of κ-Car gene mRNA expression on transgenic Kappaphycus alvarezii. Rearing of the sprout explants was conducted on semi sterile method in cone bottle with 1500 ml volume with aeration. In this study mRNA expression was analyzed in two sprouts of transgenic explant and one non- transformation control as sample. Total RNA extraction was conducted using a TRIzol ® Reagent, and complementer DNA cDNA synthesis was conducted using an Iscript TM cDNA Synthesis Kit with reverse transcriptase PCR RT-PCR technique. Expression level quantification of κ-Car gene was conducted by a quantitative real time PCR qrt-PCR method. The result of rearing culture showed that survival percentage of transgenic K. alvarezii explants was 100, transgenic candidates explant others was 36 and controls was 43. The number of shoots was 2-4 each explants, and long shoots was 5-8 mm. Results of e xpression analysis indicated the normalized mRNA κ-CarrageenaseActin gene expression level was 0.983 and 0.962 in transgenic T1 and T2, respectively. Thus,