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Production of bioethanol from biomass of agricultural waste has been hindered with a high production cost because enzymes needed for the process has to be imported with relatively a high price. Genetic engineering using its encoding genes is able to produce those enzymes with lower cost. In this report we described a research aimed to clone gene encoding β-1,6-glucanase from Trichoderma harzianum with a relatively rapid and inexpensive method, by means of RT- PCR using gene specific primers. The primers were designed based on the DNA sequence of the target gene from the same species of organism used in this research. RT-PCR using that primers resulted in DNA fragment with sizes corresponding to the predicted size of full length gene encoding β-1,6-glucanase, about 1300 bp. After a sequential experiments of cloning using pGEM-T Easy vector, DNA sequencing and BlastN - BlastX analyses of the sequences, it was proven that the isolated DNA was full length gene of β-1,6-glucanase. This was implied from the percentage of Identity and E-value which were 96% and 0.0 (< e-04) respectivety. Abstrak Produksi bioetanol dari biomassa limbah pertanian, terkendala oleh tingginya biaya produksi karena enzim yang diperlukan untuk proses tersebut masih harus diimpor dengan harga yang relatif mahal. Melalui rekayasa genetika menggunakan gen-gen penyandinya, enzim-enzim tersebut dapat diproduksi dengan biaya yang lebih murah. Penelitian ini bertujuan untuk mengklon gen penyandi β-1,6-glukanase dari Trichoderma harzianum secara cepat dan ekonomis, dengan RT-PCR menggunakan primer spesifik. Primer tersebut dirancang berdasarkan sekuen DNA dari gen target asal spesies organisme yang sama dengan yang digunakan dalam penelitian. RT-PCR dengan primer tersebut menghasilkan fragmen DNA yang ukurannya sesuai dengan gen lengkap penyandi β-1,6-glukanase, yaitu sekitar 1300 bp. Setelah secara berurutan diklon menggunakan vektor pGEM-T Easy, sekuensing urutan DNA dan analisis BlastN maupun BlastX dari sekuen yang diperoleh, terbukti bahwa fragmen DNA tersebut adalah gen lengkap penyandi β-1,6-glukanase. Hal ini ditunjukkan oleh Nilai Kesamaan (Identity) dan E-Value yang masing-masing mencapai 96% dan 0.0.

Kloning gen penyandi β-1,6-glukanase kapang secara cepat dengan teknik RT-PCR menggunakan primer spesifik Rapid cloning for gene encoding fungal β-1,6-glucanase by means of RT-PCR using specific primers