Jurnal Bioteknologi Pertanian,of Vol. 9, No. 2, transcriptional 2004, pp.

33-40 Isolation and characterization anthocyanin activator genes ...

33

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]

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

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