Kloning cDNA lengkap penyandi ACCase subunit biotin carboxylase dari mesokarp kelapa sawit (Elaeis guineensis Jacq.) Cloning of full length cDNA encoding ACCase subunit biotin carboxylase from mesocarp of oil palm (Elaeis guineensis Jacq.)

Asmini BUDIANI, Antonius SUWANTO, Hajrial ASWIDINNOO, Djoko SANTOSO, Basil J NIKOLAU

Abstract


Abstract
Acetyl-CoA Carboxylase (ACCase) is considered to be
one of the key enzymes in palm oil biosynthesis. Availability
of genes encoding this enzyme would give some advantages
in the molecular breeding of oil palm. Over expression of
the genes in the oil palm mesocarp might increase the oil
production in this tissue. On the other hand, down
regulating of ACCase could divert the central metabolite
Acetyl-CoA to other product such as PHB (Polyhydroxy-
butyrate), one of the known biodegradable plastic. This
paper reported the work of cloning of the full length coding
sequence of biotin carboxylase (BC), one subunit of the
ACCase. Based on the DNA sequence of the BC conserved
region that had cloned previously, primers pairs were
designed to amplify 5’- and 3’- cDNA ends of BC using
RACE-PCR. The RACE products of 5’- and 3’- cDNA ends
of BC were cloned into E.coli, and the DNAs were
sequenced and analysed. The full cDNA of BC was obtained
by reisolation of the cloned 5’- and 3’- cDNA ends followed
by digestion using KpnI, ligation into pGEM-T vector and
cloning into E.coli. Colony PCR was carried out to confirm
that the target gene has been cloned. The recombinant
plasmid containing full cDNA of BC was then isolated for
DNA sequencing. The results showed that the 5’-BC (1367
bp), 3’- BC (1032 bp), and the full length cDNA encoding
BC (2182 bp) had been successfully cloned, and the DNA
sequence had been confirmed as gene encoding ACCase
subunit biotin carboxylase.

Abstrak
Acetyl-CoA Carboxylase (ACCase) merupakan salah
satu enzim kunci dalam biosintesis minyak sawit. Keter-
sediaan gen penyandi enzim ini sangat berguna dalam
pemuliaan kelapa sawit secara molekuler. Over-ekspresi gen
penyandi ACCase pada mesokarp dapat meningkatkan pro-
duksi minyak pada jaringan tersebut. Sebaliknya ekspresi
ACCase dapat ditekan melalui mekanisme down regulation 

sehingga metabolit central Acetyl-CoA dapat diarahkan
untuk menghasilkan produk lain seperti PHB (polyhydro-
xybutyrate), salah satu jenis biodegradable plastik yang
telah banyak dikenal. Penelitian ini bertujuan untuk
mengklon cDNA lengkap penyandi ACCase subunit biotin
carboxylase (BC) dari mesokarp kelapa sawit. Berdasarkan
sekuen DNA daerah konservatif BC yang telah diklon dari
mesokarp kelapa sawit pada penelitian sebelumnya, dua
pasang primer dirancang untuk mengamplifikasi daerah
ujung 5’- dan 3’- cDNA BC dengan RACE-PCR. Produk
5’-RACE dan 3’-RACE diklon dan disekuen. cDNA
lengkap penyandi BC diperoleh dengan jalan mengisolasi
kembali fragmen 5’- dan 3’- cDNA terklon, dilanjutkan
dengan digesti menggunakan enzim restriksi KpnI, ligasi
kedua fragmen ke vektor kloning pGEM-T, dan introduksi
ke dalam E. coli. Setelah dilakukan PCR koloni untuk
menguji keberhasilan kloning, plasmid rekombinan yang
mengandung cDNA lengkap dari BC diisolasi untuk analisis
sekuen DNA. Dari penelitian ini fragmen cDNA 5’-BC
(1367 pb) dan 3’- BC (1032 pb), serta cDNA lengkap
penyandi BC berukuran 2182 pb telah diperoleh dan diklon
dalam E. coli. Analisis sekuen DNA mengkonfirmasi bahwa
cDNA terklon adalah benar gen penyandi ACCase subunit
biotin carboxylase.


Keywords


Heteromeric ACCase, palm oil biosynthesis, fatty acid, RACE-PCR

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DOI: http://dx.doi.org/10.22302/iribb.jur.mp.v81i2.43

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