Ekspresi gen penyandi ACCase subunit biotin karboksilase dari mesokarp buah kelapa sawit pada Escherichia coli Expression of gene encoding ACCase subunit biotin carboxylase from oil palm fruit mesocarp in Escherichia coli





Production of palm oil could be increased, one of which is by increasing oil content in the mesocarp of oil palm. This might be done by increasing the activity of key enzymes of the oil biosynthesis pathway in the oil palm mesocarp. Acetyl-CoA carboxylase has been reported as the enzyme that plays important role in oil accumulation in the oil palm mesocarp. Gene encoding one subunit of ACCase, biotin carboxylase (BC) had been isolated from oil palm mesocarp and cloned in E. coli. This reseach was aimed to examine the expression of the cloned BC gene in the E. coli. The cloned cDNA encoding BC was reisolated from recom-binant E. coli by PCR using spesific primers. The PCR products were verified in the agarose gel, and then ligated to pTrcHis-TOPO expression vector. The ligation product, recombinant vector pTrcHis-TOPO/BC, was introduced into E. coli XL1-Blue. Recombinant colonies grew in the selection media were analyzed using PCR to confirm the existent of the target DNA.  The colonies, which have been confirmed to contain target DNA were then subcultured in the LB media, for extraction of total protein. The protein extract was then analyzed quantitatively by Lowry method, and qualitatively by electrophoresis on SDS polyacrylamide gel. The result showed that recombinant plasmid pTrcHis-TOPO/BC has been successfully inserted into E. coli XL-1 Blue. SDS-PAGE analysis of the extracted protein showed that recombinant E. coli produced specific protein with MW of about 43 kDa, much higher compared with that of untransformed E. coli. This results demonstrate that  the cloned BC was strongly expressed in E. coli




Produksi minyak sawit dapat ditingkatkan, salah satu-nya dengan meningkatkan rendemen minyak. Hal ini dapat dilakukan dengan cara  meningkatkan aktivitas enzim kunci biosintesis minyak pada mesokarp buah sawit. Acetyl-CoA carboxylase telah dilaporkan merupakan enzim yang berperan penting dalam akumulasi minyak pada mesokarp kelapa sawit. Pada penelitian sebelumnya, gen penyandi salah satu subunit ACCase, yaitu biotin carboxylase (BC), telah diisolasi dari jaringan mesokarp kelapa sawit dan diklon pada E.coli. Tujuan penelitian ini adalah untuk menguji ekspresi gen tersebut pada E. coli. cDNA penyandi BC diisolasi kembali dari E. coli rekombinan dengan PCR menggunakan pasangan primer spesifik. Hasil isolasi diveri-fikasi pada gel agarose, kemudian diligasikan dengan vektor ekspresi  pTrcHis-TOPO.  Vektor  rekombinan (pTrcHis-TOPO/BC) hasil ligasi diintroduksikan ke dalam E. coli XL1-Blue. Koloni rekombinan yang tumbuh pada media seleksi dianalisis menggunakan PCR untuk mengkonfirmasi ada tidaknya sisipan DNA target. Koloni yang  terbukti mengandung sisipan DNA target dikulturkan pada media LB kemudian protein total diekstrak dari kultur E. coli dan dianalisis dengan elektroforesis SDS-PAGE. Hasil PCR koloni menunjukkan bahwa transformasi E. Coli XL-1 Blue menggunakan konstruk vektor rekombinan pTrcHis-TOPO/ BC berhasil baik. Analisis SDS-PAGE dari ekstrak protein menunjukkan bahwa E. coli rekombinan menghasilkan protein dengan berat molekul sekitar 43 kDa yang inten-sitasnya jauh lebih tinggi dibandingkan dengan protein yang sama yang dihasilkan oleh E. coli  yang tidak ditrans-formasi. Hal ini membuktikan bahwa gen penyandi BC dalam vektor pTrcHis-TOPO dapat diekspresikan dengan kuat pada   E. coli.


Palm oil, oil biosynthesis, over expression, acetyl-CoA carboxylase

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DOI: http://dx.doi.org/10.22302/iribb.jur.mp.v82i1.29

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