Cloning and characterization of OSB1 gene controlling anthocyanin biosynthesis from Thai black rice

OSB1 gene encodes myc-type basic helix-loop-helix (bHLH) transcription factor which controls expression of several structural genes involving in anthocyanin biosynthesis in rice. In this study, the expression of OSB1 gene was investigated in young leaves and developing seeds of six rice varieties, including white (Taichung 65 and Sasanishiki), red (Sang Yod and Hom Mali Dang) and black (Khum and Lerm Poa), by RT-PCR. The OSB1 gene was expressed in both leaves and seeds of all six rice varieties. The full-length coding sequences of OSB1 genes were isolated from young leaves of white rice Sasanishiki and black rice Khum. Nucleotide sequence analysis revealed that Khum and Sasanishiki had open reading frames (ORF) of 1,767 and 1,725 bp, respectively which were 99–100% and 100% identical with OSB1 genes in black and white rice reported in GenBank database, respectively. It was found that OSB1 gene from white rice showed a 2-bp insertion in the 7 th exon that caused frameshift mutation and premature termination, leading to the truncation of 14 amino acids at C-terminus of the regulatory protein. Moreover, the amino acid substitution of T64M was found in white rice OSB1 protein, affecting the conserved N-terminal interacting domain, probably causing the non-function of the OSB1 gene in white rice. The OSB1 gene from black rice was active in anthocyanin biosynthesis, suggesting this gene might play an important role in anthocyanin pigmentation, especially in seeds. The cloned OSB1 genes will be further analyzed for gene functions to understand the regulation of anthocyanin biosynthesis in colored rice.