Isolasi Gen Palmitoyl-ACP Thioesterase pada kelapa sawit (Elaeis guineensis Jacq.)

SUKARIAWAN, Aries, Dr.Ir. Aziz Purwantoro, M.Sc

2005 | Tesis | S2 Agronomi

Peningkatan kualitas tanaman sebagai penghasil minyak menjadi sasaran pemuliaan tanaman kelapa sawit, salah satunya melalui rekayasa genetika. Gen palmitoyl-ACP thioesterase (PAT) merupakan penyandi enzim yang berperan dalam sintesis asam palmitat yang keberadaan asam lemak jenuh ini masih berlebihan dan kurang diinginkan dalam crude palm oil (CPO), karena potensi yang merugikan kesehatan. Langkah awal yang dilakukan dalam upaya rekayasa genetika adalah mengisolasi gen yang terkait dengan negative trait tanaman tersebut. Melalui gene silencing, diharapkan dapat menghambat ekspresi gen PAT sehingga mengurangi proporsi asam palmitat dan meningkatkan potensi kandungan asam oleat yang diharapkan dalam CPO. Tujuan dalam penelitian ini adalah memperoleh sekuen nukleotida gen PAT dari tanaman kelapa sawit. Pelaksanaan penelitian meliputi ekstraksi DNA dari daun, isolasi gen melalui disain primer spesifik dan amplifikasi, kloning gen PAT meliputi ligasi dan transformasi, serangkaian konfirmasi dan verifikasi serta sekuensing gen PAT. Hasil penelitian menunjukkan bahwa telah diisolasi 2 fragmen DNA menggunakan 2 pasang primer yang berbeda yaitu fragmen PAT I dan PAT II. Fragmen gen PAT I teramplifikasi menggunakan primer PAT 2 dan PAT TF. Melalui kloning yaitu ligasi dan transformasi dalam sel Escherichia coli strain DH5α, telah dihasilkan plasmid rekombinan yang membawa fragmen gen PAT I. Fragmen target dalam plasmid inilah yang kemudian disekuen dan menghasilkan fragmen sebesar 1.063 bp. Fragmen gen PAT II dengan panjang sekitar 750 bp, teramplifikasi menggunakan primer PAT U dan PAT UR.

Plant quality improvement, as a source of oil, is one of the objective of oil palm plant breeding program, such as genetic engineering. Palmitoyl-ACP thioesterase (PAT) gene is an enzyme code, which influenced the synthesis of palmitic acid, where its existence is unexpected in crude palm oil (CPO), because of its negative impact on human health. The first step in genetic engineering is isolating the gene which related to the plant’s negative trait. The gene silencing phase is expected to avoid the expression of gene PAT, to reduce the proportion of palmitic acid as well as improving the content of oleic acid, which is expected to present in CPO. The aim of the research was to have nucleotide sequence of the PAT gene of the oil palm. The procedure of the research included, extraction the DNA from leaves, gene isolation through specific primer design and amplification, cloning the PAT gene including ligation and transformation, a series of confirmation and verification, and PAT gene sequencing. The result showed that 2 fragments of DNA has been isolated by using 2 different pair of primers, they are PAT I and PAT II fragment. PAT I gene fragment was amplified by using PAT 2 and PAT TF primers. Recombinant plasmid carrying PAT I gene fragment has been produced through cloning, ligation and transformation inside the strain of DH5α Escherichia coli. The targeted fragment inside the plasmid then was sequence and producing 1,063 bp fragment. 750 bp length PAT II gene, was amplified by PAT U and PAT UR primer.

Kata Kunci : Gen palmitoyl-ACP thioesterase, Elaeis guineensis Jacq., Isolasi, kloning dan sekuensing, Palmitoyl-ACP thioesterase gene, Elaeis guineensis Jacq., isolation, cloning, and sequencing.