Isolation and characterization of protein differentially expressed during oil palm mesocarp development Isolasi dan karakteristik protein terekpresi secara diferensial selama perkembangan mesokarp pada kelapa sawit

A BUDIANI, D SANTOSO, H ASWINDINNOOR, A SUWANTO, S SUDIATSO

Abstract


Ringkasan

Pada kelapa sawit, mesokarp merupakan jaringan yang lebih dikhususkan untuk mensintesis minyak. Akumulasi minyak pada jaringan ini terjadi selama perkembangan buah. Beberapa enzim yang terlibat dalam biosintesis minyak tampaknya disintesis hanya pada periode tertentu dari biosintesis minyak. Sedangkan protein regulator diduga ada pada saat minyak mulai disintesis atau beberapa saat sebelumnya. Sebagai bagian dari usaha mengklon gen kunci untuk biosintesis minyak, penelitian ini bertujuan mengidentifikasi dan mengisolasi protein yang terekspresi secara diferensial sesuai perkembangan buah. Sebagai bahan penelitian digunakan jaringan mesokarp dari berbagai umur buah sawit. Untuk setiap fase perkembangan buah dilakukan analisis kandungan minyak dan protein total. Elektroforesis gel poliakrilamid-SDS (SDS – PAGE) dan elektroforesis dua dimensi (2-D) digunakan untuk mempelajari dan mendeteksi adanya pita protein spesifik yang terekspresi secara diferensial sejalan dengan peningkatan kandungan minyaknya. Hasil penelitian menunjukkan bahwa minyak mulai aktif disintesis pada saat buah berumur 17 minggu setelah antesis. Konsentrasi protein total tidak meningkat sejalan dengan peningkatan kandungan minyaknya. Dari hasil SDS-PAGE terdeteksi dua protein, yaitu protein dengan berat molekul (BM) 31,0 kDa dan 34,3 kDa yang meningkat ekspresinya pada awal dan menjelang periode aktif sintesis minyak. Analisis lebih lanjut dengan elektroforesis 2-D menunjukkan bahwa protein 31,0 kDa terdiri dari dua protein dengan pI 4,64 dan pI 4,95, sedangkan protein 34,3 kDa merupakan protein tunggal dengan pI 4,56. Sikuensing secara parsial kedua protein tersebut menunjukkan adanya dua polipeptida dari protein 31,0 kDa yang mempunyai homologi tinggi dengan subunit biotin karboksilase ht-ACCase, dan empat polipeptida yang mempunyai homologi dengan enoilACP reduktase. Sedangkan protein 34,3 kDa mempunyai homologi dengan gliseraldehida 3-fosfat dehidrogenase.

 

Summary

In oil palm, mesocarp is tissue specialized for oil synthesis. Accumulation of oil in this tissue occurs during fruit development. It is likely that some enzymes involved in oil biosynthesis are synthesized only in a certain period of oil biosynthesis, while regulatory proteins may present at the beginning or right before the period of active oil synthesis. As a part of research work on cloning of gene encoding key enzymes for oil biosynthesis in palm mesocarp, this research was aimed to identify and isolate proteins differentially expressed during fruit development. Mesocarps from different developmental stage of fruit were used for analysis of oil content and protein concentra-tion. Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and two dimentional (2-D) electrophoresis were used to study and detect specific protein bands differentially expressed during fruit development. It was shown that oil synthesis was started at 17 weeks after anthesis (WAA). There was no correlation between concentrations of total protein with oil content during mesocarp development. From the SDS-PAGE, two protein of 31.0 kDa and 34.3 kDa were detected that their expression increased at the beginning and just before the period of active oil biosynthesis respectively. Further analysis with 2-D electrophoresis showed that 31.0 kDa-protein consist of two proteins, with pI 4,64 and pI 4,95, while 34.3 kDa protein is a single protein with pI 4,56. Partial amino acid sequencing data of the 31.0 kD protein showed that two polypeptides highly homologous with ht-ACCase biotin carboxylase subunit and four polypeptides homologuus with enoyl-ACP reductase, whereas 34.3 kD protein showed homology with glyceraldehyde-3 phosphate dehydrogenase.


Keywords


Marker protein, oil biosynthesis, oil-palm mesocarp

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DOI: http://dx.doi.org/10.22302/iribb.jur.mp.v70i1.130

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