Isolasi Fragmen Gen Penyandi Invertase Beberapa Varietas Tebu (Saccharum officinarum L.)

Abstract

Tebu (Saccharum officinarum L.) merupakan tanaman tropis dan subtropis yang dapat mengakumulasi sukrosa hingga 60% bobot kering dengan rata-rata akumulasi pada varietas komersil berkisar antara 350-400 mg/g bobot kering dan maksimal hingga 600 mg/g bobot kering batang tebu dewasa. Sukrosa merupakan produk utama tanaman tebu yang memiliki nilai ekonomi. Upaya peningkatan akumulasi sukrosa dalam tebu dapat dilakukan dengan pendekatan pemuliaan tanaman. Salah satu teknik pemuliaan yang dapat digunakan adalah modifikasi genetik pada enzim invertase yang terlibat langsung dalam akumulasi sukrosa. Enzim yang berperan dalam metabolisme sukrosa yaitu enzim sukrosa-fosfat sintase dan sukrosa fosfatase dalam proses sintesis sukrosa, enzim sukrosa sintase dan invertase dalam proses perombakan sukrosa. Enzim invertase merupakan enzim hidrolase yang memotong sukrosa menjadi monosakarida yaitu gula heksosa berupa glukosa dan fruktosa. Pola aktivitas ketiga enzim invertase pada jaringan penyimpanan berhubungan dengan laju pertumbuhan tebu dan kandungan sukrosa dalam tebu. Teknik modifikasi genetik yang pernah dilakukan adalah teknik antisense untuk down regulation ekspresi gen invertase. Langkah awal dalam proses modifikasi genetik adalah mengetahui sekuen nukleotida penyandi enzim invertase. Berdasarkan latar belakang tersebut, tujuan penelitian ini adalah mengisolasi dan mengarakterisasi gen penyandi invertase dari tiga varietas Indonesia serta memprediksi struktur 3D protein enzim invertase. Penelitian dilakukan melalui beberapa tahap yaitu penanaman tebu varietas Bulu Lawang (BL), PS 862, dan Kidang Kencana (KK), induksi kalus ketiga varietas, ekstraksi RNA total, sintesis cDNA, amplifikasi gen invertase, dan analisis bioinformatika yang meliputi analisis hubungan filogenetik invertase, fisiokimia protein invertase, serta prediksi struktur 3D protein invertase. Penelitian ini berhasil mengamplifikasi invertase dari ketiga varietas tebu. Berdasarkan analisis hubungan filogenetik, sekuen gen invertase dari varietas BL (CWINV7_BL) dan PS 862 (CWINV7_PS862) merupakan kelompok invertase dinding sel, invertase dari varietas KK (NAINV6_KK) merupakan kelompok invertase netral/alkali. Panjang sekuen asam amino CWINV7_BL yaitu 407 asam amino, CWINV7_PS862 331 asam amino, dan NAINV6_ KK 358 asam amino. Berdasarkan analisis struktur 3D protein invertase, ditemukan kemiripan struktur dengan kristalografi enzim dari kelompok -fruktofuranosidase, CWINV7_BL dan CWINV7_PS862 memiliki ligan berupa sukrosa dengan residu sisi aktif berupa asam aspartat dan asam glutamat. Ketiga invertase termasuk dalam famili glikosida hidrolase, memiliki enam motif sekuen, serta memiliki peran dalam respon tanaman terhadap cekaman

Sugarcane (Saccharum officinarum L.) is a tropical as well as a subtropical crop. Sugarcane can accumulate sucrose up to 60% of the plant’s dry weight with average sucrose content comercially range to 350-400 mg/g and maximum up to 600 mg/g of plant’s dry weight. Sucrose is sugarcane’s main product and has high economic value. Improving sucrose accumulation in sugarcane can be done by plant breeding programs such as genetic modification. The key regulatory enzymes like sucrose phosphate synthase, sucrose phosphatase, sucrose synthase, and invertases have been used as target to develop high sucrose sugarcane cultivars in the genetic modification programs. Invertases catalyze the irreversible hydrolysis of hexoses such as sucrose into glucose and fructose. Invertases contribute to plant growth and regulate sucrose accumulation. Many efforts have been made to control invertases activity in sugarcane through down regulation methods. The first key step before accomplished genetic modification is to obtain invertases gene sequences in order to understand the physiological roles and metabolic mechanism regulated by invertases. Based on this background, the objectives of this research are to isolate invertase gene sequences from three Indonesia’s varieties, to characterize and to predict 3D structure of invertases. This research was conducted through several stages consisting of replanting of sugarcane Bulu Lawang (BL) variety, PS 862 variety, and Kidang Kencana (KK) variety, callus induction, total RNA extraction, cDNA synthesis, amplification of invertase gene, and bioinformatics analysis. Bioinformatics analysis include phylogenetic analysis, analysis of invertases’ physicochemical properties, and 3D predicted structures of invertases. In this research, invertase gene family were isolated from three different cultivars. As the results of phylogenetic analysis, invertase genes from BL cultivar (CWINV7_BL) and PS 862 cultivar (CWINV7_PS862) are indicated as members of cell wall invertases group, whereas invertase gene from KK cultivar (NAINV6_KK) is indicated as members of neutral/alkaline invertase. The amino acid sequences of invertases have length of 407 amino acids for CWINV7_BL, 331 amino acids for CWINV7_PS862, and 358 amino acids for NAINV6_KK. 3D predicted structures from CWINV7_BL and CWINV7_PS862 are similar to crystallography structure of -fructofuranosidase. Both structures have sucrose as the ligand with aspartic acid and glutamic acid as active-site amino acid residues. Invertase from Indonesian sugarcane cultivars were from superfamily of glycoside hydrolase and may exert essential roles in stress-related responses. All invertases has six conserved motifs which related to biological function of invertase.