Karakterisasi Ekson-Intron 1, 5, dan 6 Gen Endo-β-1,4-glukanase pada Rayap Coptotermes curvignathus
Characterization of Exon-Intron 1, 5, 6 Endo-β-1,4-glucanase Genes of Termites Coptotermes curvignathus.
Abstract
An agricultural waste with cellulose-containing can be processed into ethanol. Conversion of cellulose into bioethanol requires cellulase enzyme. Cellulase enzyme from termite is able to degrade cellulose. Termite Coptotermes curvignathus is able to degrade cellulose due to cellulase enzymes encoded by genes endo-β-1,4-glucanase. Gene sequences of endo-β-1 ,4-glucanase termite C. curvignathus (CcEG) resulted from previous research has not completed yet, hence this research was aimed to complete the gene characterization. Method that used in this study were CTAB DNA extraction with modified method, DNA amplification, and gene sequencing. The size of exons 1, 5, and 6 CcEG were 46, 79, and 163 bp respectively and the size of intron 1, 5, and 6 were 483, 725, and 274 bp, respectively. The exon 5 CcEG gave further contribution to the previous research, with 26 bp of overlap bases. Exon 1, 5, and 6 CcEG showed similar size to C. formosanus (CfEG) each of which were 46, 178, and 163 bp, respectively. GC composition was 51.4% in exons, while the introns possessed 42,3% of GC. Intron 1, 5, and 6 CcEG were commenced with GT and ended with AG. Amino acid deduction analysis of exon 1, 5, and 6 CcEG resulted 95 putative amino acids with methionine as the first amino acids and the protein was classified in a group of Glycosyl Hydrolase Family 9 (GHF9). BLAST and genetic distance analysis of CcEG with C. formosanus (CfEG), Reticulitermes speratus (RsEG), and Nasutitermes takasagoensis (NtEG) showed high level of similarity with CfEG. The results of endo-β-1,4-glucanase gene characterization of C. curvignathus were expected as basic data for further in vitro cellulase enzyme studies. Limbah pertanian yang mengandung selulosa dapat diolah menjadi bioetanol. Konversi selulosa menjadi bioetanol memerlukan enzim selulase. Selulase yang dihasilkan rayap mampu mendegradasi selulosa. Rayap Coptotermes curvignathus mampu mendegradasi selulosa karena enzim selulase yang disandikan oleh gen endo-β-1,4-glukanase. Sekuen gen endo-β-1,4-glukanase rayap C. curvignathus (CcEG) dari penelitian sebelumnya belum lengkap dikarakterisasi, sehingga penelitian ini bertujuan melengkapi karakterisasi gen endo-β-1,4-glukanase pada rayap C. curvignathus. Metode yang digunakan adalah ekstraksi DNA menggunakan metode CTAB yang dimodifikasi, amplifikasi DNA, dan sekuen DNA. Hasil amplifikasi ekson 1, 5, 6 CcEG berukuran 46, 79, dan 163 pb, sedangkan intron 1, 5, dan 6 secara berurutan sebesar 483, 725, dan 274 pb. Sekuen DNA Ekson 5 CcEG hasil penelitian ini melengkapi penelitian sebelumnya dengan jumlah nukleotida yang berada pada posisi yang sama (overlap) sebanyak 26 pb. Ukuran panjang ekson 1, 5, dan 6 CcEG sama dengan C. formosanus (CfEG) masing-masing yaitu 46, 178, dan 163 pb. Komposisi GC ekson CcEG sebesar 50.3% pada ekson sedangkan pada intron sebesar 43.3%. Intron 1, 5, dan 6 CcEG diawali dengan basa GT dan diakhiri AG. Hasil analisis deduksi gen endo-β-1,4-glukanase ekson 1, 5, dan 6 CcEG menghasilkan 94 asam amino putative dengan metionin sebagai awal asam amino, dan termasuk kelompok Glycosyl Hydrolase Family 9 (GHF9). Analisis BLAST dan jarak genetik CcEG terhadap C. formosanus (CfEG), Reticulitermes speratus (RsEG), dan Nasutitermes takasagoensis (NtEG) menunjukkan tingkat kekerabatan yang tinggi dengan CfEG. Hasil karakterisasi gen endo-β-1,4-glukanase pada rayap C. curvignathus.diharapkan dapat menjadi informasi dasar penelitian in vitro karakterisasi enzim selulase rayap tersebut.
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