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33

Jurnal Bioteknologi
Pertanian,ofVol.
9, No. 2, transcriptional
2004, pp. 33-40activator genes ...
Isolation
and characterization
anthocyanin

Isolation and characterization of anthocyanin transcriptional activator


genes from cDNA library of sweet potato
Isolasi dan karakterisasi gen pengaktif transkripsi antosianin dari pustaka cDNA ubi jalar
Muchdar Soedarjo1 and Koshun Ishiki2
Indonesian Legume and Tuber Crops Research Institute, PO Box 66, Malang 65101, Indonesia,
E-mail:
muchdar_soedarjo@yahoo.com
2
Japan International Research Center for Agricultural Sciences, Okinawa Subtropical Station, Maezato 1091-1, Ishigaki,
Okinawa, 907-0002, Japan, E-mail: ishiki@jircas.affrc.go.jp
1

ABSTRAK
Biosintesis antosianin secara temporal dan spasial pada
jaringan tanaman dikendalikan oleh gen pengaktif transkripsi
antosianin (GPTA). Peran GPTA sebagai pengaktif biosintesis
antosianin belum dikaji secara intensif pada tanaman ubi jalar.
Suatu kajian dilaksanakan untuk mengisolasi dan mengkarakterisasi GPTA dari jaringan umbi ubi jalar berwarna ungu.
GPTA tipe myb dari Perilla frutescens digunakan sebagai probe
untuk mengisolasi GPTA dari pustaka cDNA daging umbi ubi
jalar berwarna ungu. Dua dari 14 klon menunjukkan kesamaan
dengan gen pengaktif transkripsi tipe myb, termasuk GPTA
dari jagung. Dua klon tersebut merupakan klon yang sama
karena mempunyai urutan basa yang sama. Klon yang diperoleh diberi nama sp4 dan berukuran 1253 bp. Pengkodean
urutan tentatif dimulai dari nt. 97 sampai nt. 933, sehingga
klon sp4 mempunyai 278 residu asam amino. Klon sp4 mengandung dua DNA binding domain pada terminal N dan
domain asam pada carboxyl terminal. Fitur ini umumnya
dijumpai pada GPTA tipe myb. Oleh karena itu, klon sp4
diperkirakan berperan sebagai gen pengaktif transkripsi
antosianin pada ubi jalar.
[Kata kunci: Ubi jalar, antosianin, gen pengaktif transkripsi,
pustaka cDNA]

ABSTRACT
Temporal and spatial biosyntheses of anthocyanin in various
plant tissues are coordinately controlled by the expression of
anthocyanin transcriptional activator genes (ATAG). So far,
ATAG has not been extensively studied on sweet potato in
spite of its high anthocyanin content. A study was undertaken
to isolate and characterize the ATAG from the purple-fleshed
tuberous root of sweet potato. The myb type ATAG fragment
of Perilla frutescens (ao-jiso) was used as a probe to isolate
the myb type ATAG from the cDNA library of the purplefleshed tuberous root of sweet potato. Fourteen positive
plaques were detected from the cDNA library. Two clones out
of the 14 clones revealed homology to the myb type transcriptional activator gene, including the myb type ATAG in
maize. The nucleotide sequence of the two clones were the
same, hence, they belonged to the same clone. The clone was
named as sp4 and had a size of 1253 bp. Putative coding
sequence started from nt. 97 to nt. 933. Thus the clone had

278 amino acid residues. The clone contained two DNA


binding domain at its N terminal and acidic region at carboxyl
terminal, which were the features commonly observed in the
myb type ATAG. Therefore, the clone was probably a transcriptional regulatory gene of anthocyanin biosynthesis in
sweet potato.
[Keywords: Sweet potatoes, anthocyanins, transcriptional
activator gene, cDNA library]

INTRODUCTION
Temporal and spatial biosyntheses of anthocyanin in
various plant tissues are coordinately controlled by
the expression of anthocyanin transcriptional activator genes (ATAG) (Ludwig et al. 1989; Goff et al.
1992; Radicella et al. 1992; Hu et al. 1996; Sainz et al.
1997; Bradley et al. 1998). ATAG falls into two classes,
myb and myc types. The product of these two ATAG
types interact each other to regulate the expression of
anthocyanin structural genes (Grotewold et al. 2000).
The myb type anthocyanin transcriptional activator is
featured by the presence of the DNA binding domain
at the N terminal and acidic region at the carboxyl
terminal, and the myc type contains a basic helix-loophelix region (Ludwig and Wessler 1990; Gong et al.
1999a and 1999b).
ATAG studies have been extensively held on several
plant species such as maize, rice, petunia, snapdragon, and Perilla (Goodrich et al. 1992; Lloyd et al.
1992; Quatrocchio et al. 1993; Gong et al. 1999a and
1999b, Spelt et al. 2000). Previous investigators (Shi
et al. 1992; Yoshimoto et al. 1999; Yoshinaga et al.
1999) reported that anthocyanin was also found at
high concentration in the purple-fleshed tuberous
root. However, the studies of ATAG on sweet potato
have not been extensively undertaken. Liu (2000)
initially studied the ATAG on sweet potato by
employing the AFLP. A primer designed from the
conserved sequence of the myb type ATAG and