EKSPRESI GEN RFT1 DALAM SIKLUS DIURNAL MASA TRANSISI PEMBUNGAAN TANAMAN PADI HITAM (Oryza sativa L. “Cempo Ireng”)

FARIDA NUR MALIKA, Dr. Yekti Asih Purwestri, S.Si., M.Si.

2014 | Skripsi | BIOLOGI

Di Indonesia, terdapat banyak kultivar padi lokal, salah satunya yaitu padi hitam kultivar Cempo Ireng. Pada padi, terdapat 13 gen FT-like yang telah teridentifikasi (OsFTL1-OsFTL13). OsFTL3/RFT1 adalah salah satu gen yang paling aktif berperan dalam menginduksi pembungaan. Penelitian ekspresi gen RFT1 padi hitam yang telah dilakukan menunjukkan ekspresi temporal gen RFT1 pada padi hitam paling jelas pada 70-75 HST (Hari Setelah Tanam). Penelitian ini dilakukan untuk mengetahui waktu ekspresi gen RFT1 pada siklus harian/siklus diurnal selama masa transisi pembungaan. Metode penelitian meliputi isolasi RNA, uji kualitas RNA, pembuatan cDNA, amplifikasi gen RFT1, dan kuantifikasi ekspresi gen RFT1. Isolasi RNA dari daun padi hitam umur 70 dan 90 HST masing-masing pada pukul 08.00, 12.00, dan 16.00 dilakukan menggunakan metode RNeasy Plant Mini Kit. Uji kualitas RNA dilakukan dengan spektrofotometer pada λ = 260 nm dan 280 nm. Pembuatan cDNA dilakukan dengan two-step RT-PCR dan cDNA dicek kualitasnya dengan amplifikasi gen Ubiquitin sebagai kontrol internal. Hasil amplifikasi Ubiquitin dan RFT1 divisualisasi dengan elektroforesis gel agarose. Kuantifikasi ekspresi gen RFT1 dan Ubiquitin dilakukan menggunakan program ImageJ untuk mengestimasi konsentrasi RFT1 dan Ubiquitin berdasarkan ketebalan dan densitas pita pada hasil elektroforesis. Hasil penentuan kualitas RNA menunjukkan RNA padi hitam memiliki kualitas yang bagus dengan rasio 1,9-2,3. Hasil elektroforesis menunjukkan gen Ubiquitin terekspresi pada semua sampel dengan ukuran sekitar 200 bp. Ekspresi gen RFT1 padi hitam terlihat pada semua sampel dengan ukuran sekitar 200 bp. Tingkat ekspresi RFT1 pada pukul 08.00 berkisar 2,5-3 kali lebih tinggi daripada ekspresi pada pukul 12.00 dan 16.00.

In Indonesia, there are a lot of local rice cultivars, one of them is black rice cultivar Cempo Ireng. In rice, there are 13 FT-like genes that have been identified (OsFTL1-OsFTL13). OsFTL3/RFT1 is one of the most active gene involved in flowering induction of rice. RFT1 gene expression of black rice that have been evaluated before, showed that temporal expression of RFT1 was most clearly viewed at 70 until 75 days after planting (DAP). The objective of this research was to evaluate the RFT1 gene expression in the daily/diurnal cycle during the flowering transition phase. Research method consists of RNA isolation, determination of RNA quality, cDNA formation, RFT1 gene amplification, and RFT1 gene expression’s quantification. RNA was isolated from black rice leaf of 70 and 90 DAP, taken at 08.00, 12.00 and 16.00 o’clock. Sample was isolated using RNeasy Plant Mini Kit. RNA quality was determined by spectrophotometer at λ = 260 nm and 280 nm. The cDNA was synthetized by two-step RT-PCR and cDNA quality was checked by Ubiquitin gene amplification as the internal control. The result of Ubiquitin and RFT1 amplification were visualized using agarose gel electrophoresis. Quantification of RFT1 and Ubiquitin expression were conducted using ImageJ program to estimate RFT1 and Ubiquitin concentration based on the width and density of bands on the electrophoresis result. The RNA had a good quality with the ratio of 1.9-2.3. Electrophoresis result showed that Ubiquitin gene was expressed in all samples with approximately 200 bp in size. RFT1 gene of black rice was expressed in all samples with approximately 200 bp in size. RFT1 expression level at 08.00 o’clock was about 2.5-3 times higher than the expression at 12.00 and 16.00 o’clock.

Kata Kunci : Padi Hitam, gen RFT1, RT-PCR