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Anthocyanins are important antioxidants found in black pericarp rice. The OsB2 gene encodes a transcription factor which can activate anthocyanin biosynthesis in rice. In this study, transformation of Kasalath variety (red pericarp rice) using OsB2 cloned from a black pericarp rice via Agrobacterium was investigated for functional study on anthocyanin biosynthesis in rice. Fifty-nine transformants were regenerated and PCR analysis showed that 84.28 % carried the OsB2 transgene. The phenotypic traits of transgenic rice were similar to those of wild-type but developing and mature seeds showed purple pigmentation on their pericarp. Segregation analysis of gus genes showed the 3 : 1 ratio in T1 generation, indicating a single gene insertion in the rice genome. Phenotypic traits of T1 progeny were similar to those of the T0 transgenic rice plants. Semi-quantitative RT-PCR analysis revealed that overexpression of OsB2 gene in seedlings was found in T1 plants. In addition, seeds of T1 transgenic rice contained higher antioxidant activity than that of the untransformed plants.
Keywords: Oryza sativa; OsB2; transformation; anthocyanin
Cossins, E., Lee, R. and Packer, L., 1998, ESR studies of vitamin C regeneration, order of reactivity of natural source phytochemical preparations, IUBMB Life 45: 583-597.
Choudhury, B.I., Khan, M.L. and Dayanandan, S., 2014, Patterns of nucleotide diversity and phenotypes of two domestication related genes (OsC1 and Wx) in indigenous rice varieties in northeast India, BMC Genet. 15: 71.
Endo, S., Sugita, K., Sakai, M., Tanaka, H. and Ebinuma, H., 2002, Single-step trans formation for generating marker-free transgenic rice using the ipt-type MAT vector system, Plant J. 30: 115-122.
Furukawa, T., Maekawa, M., Oki, T., Suda, I., Iida, S., Shimada, H. and Kadowaki, K. I., 2007, The Rc and Rd genes are involved in proanthocyanidin synthesis in rice pericarp, Plant J. 49: 91-102.
Geekiyanage, H., Jicha, G.A., Nelson, P.T. and Chan, C., 2012, Blood serum miRNA: non-invasive biomarkers for Alzheimer's disease, Exp. Neurol. 235: 491-496.
Hu, J., Anderson, B. and Wessler, S.R., 1996, Isolation and characterization of rice R genes: Evidence for distinct evolutionary paths in rice and maize, Genetics 142: 1021-1031.
Hwang, S. and Kim, Y., 2000, A simple and reliable method for preparation of cross-contamination-free plant genomic DNA for PCR-based detection of transgenes, J. Biochem. Mol. Biol. 33: 537-540.
Inta, P., Phongburaphat, W., Pongjaroenkit, S., Chowpongpang, S., Sangtong, V. and Sakulsingharoj, C., 2013, Cloning and characterization of OSB2 gene controlling anthocyanin biosynthesis in rice, Thai J. Genet. 6(1): 25-29.
Jefferson, R., 1987, Assaying chimeric genes in plants: The GUS gene fusion system, Plant Mol. Biol. Rep. 5: 387-405.
Kawahigashi, H., Hirose, S., Iwai, T., Ohashi, Y., Sakamoto, W., Maekawa, M. and Ohkawa, Y., 2007, Chemically induced expression of rice OSB2 under the control of the OsPR1, 1 promoter confers increased anthocyanin accumulation in transgenic rice, J. Agric. Food Chem. 55: 1241-1247.
Kim, D.H., Park, S., Lee, J.Y., Ha, S.H., Lee, J.G. and Lim, S.H., 2018, A rice B-Box protein, OsBBX14, finely regulates anthocyanin biosynthesis in rice, Int. J. Mol. Sci. 19: 2190.
Lepiniec, L., Debeaujon, I., Routaboul, J.M., Baudry, A., Pourcel, L., Nesi, N. and Caboche, M., 2006, Genetics and biochemistry of seed flavonoids, Ann. Rev. Plant Biol. 57: 405-430.
Lim, S.H. and Ha, S.H., 2013, Marker development for the identification of rice seed color, Plant Biotechnol. Rep. 7: 391-398.
Maeda, H., Yamaguchi, T., Omoteno, M., Takarada, T., Fujita, K., Murata, K. and Mukaino, N., 2014, Genetic dissection of black grain rice by the development of a near isogenic line, Breeding Sci. 64: 134-141.
Nagao, S. and Takahashi, M.E., 1963, Trial construction of twelve linkage groups in Japanese rice: Genetical studies on rice plant, J. Fac. Agric. Hokkaido Univ. 53: 72-130.
Na Rachasima, L., Sukkasem, R., Pongjaroen kit, S., Sangtong, V., Chowpongpang, S., and Sakulsingharoj, C., 2017, Expression analysis and nucleotide variation of OsC1 gene associated with anthocyanin pigmentation in rice, Genomics Genet. 10: 46-53.
Oikawa, T., Maeda, H., Oguchi, T., Yamaguchi, T., Tanabe, N., Ebana, K. and Izawa, T., 2015, The birth of a black rice gene and its local spread by introgression, Plant Cell 27: 2401-2414.
Oshima, M., Taniguchi, Y., Akasaka, M., Abe, K., Ichikawa, H., Tabei, Y. and Tanaka, J., 2019, Development of a visible marker trait based on leaf sheath-specific anthocyanin pigmentation applicable to various genotypes in rice, Breeding Sci., Article ID: 18151, 11 p.
Rahman, M.M., Lee, K.E. and Kang, S.G., 2016, Allelic gene interaction and anthocyanin biosynthesis of purple pericarp trait for yield improvement in black rice, J. Life Sci. 26: 727-736.
Reddy, V.S., Goud, K.V., Sharma, R. and Reddy, A.R., 1994, Ultraviolet-B-responsive anthocyanin production in a rice cultivar is associated with a specific phase of phenylalanine ammonia lyase biosynthesis, Plant Physiol. 105: 1059-1066.
Saika, H., Sakamoto, W., Maekawa, M. and Toki, S., 2011, Highly efficient visual selection of transgenic rice plants using green fluorescent protein or anthocyanin synthetic genes, Plant Biotechnol. 28: 107-110.
Saitoh, K., Onishi, K., Mikami, I., Thidar, K. and Sano, Y., 2004, Allelic diversification at the C (OsC1) locus of wild and cultivated rice, Genetics 168: 997-1007.
Sakamoto, W., Ohmori, T., Kageyama, K., Miyazaki, C., Saito, A., Murata, M., Noda, K. and Maekawa, M., 2001, The Purple leaf (Pl) locus of rice: The Plw allele has a complex organization and includes two genes encoding basic helix-loop-helix proteins involved in anthocyanin biosynthesis, Plant Cell Physiol. 42: 982-991.
Sakulsingharoj, C., Inta, P., Sukkasem, R., Pongjaroenkit, S., Chowpongpang, S. and Sangtong, V., 2014, Overexpression of OSB2 gene in transgenic rice up-regulated expression of structural genes in anthocyanin biosynthesis pathway, Thai J. Genet. 7(3): 173-182.
Sakulsingharoj, C., Phanlumpak, K., Inta, P., Pongjaroenkit, S. and Sangtong, V., 2015, Transformation of rice (Oryza sativa) cultivar Taichung 65 mediated by Agrobacterium tumefaciens, pp. 189-196, In Biology Education and Research in a Changing Planet, Springer, Singapore.
Sakulsingharoj, C., Inta, P., Sukkasem, R., Pongjaroenkit, S., Chowpongpang, S. and Sangtong, V., 2016, Cloning and characterization of OSB1 gene controlling anthocyanin biosynthesis from Thai black rice, Genomics Genet. 9: 7-18.
Shao, Y., Xu, F., Sun, X., Bao, J. and Beta, T., 2014, Phenolic acids, anthocyanins, and antioxidant capacity in rice (Oryza sativa L.) grains at four stages of development after flowering, Food Chem. 143: 90-96.
Shi, J., Yu, J., Pohorly, J.E. and Kakuda, Y., 2003, Polyphenolics in grape seeds biochemistry and functionality, J. Med. Food 6: 291-299.
Shin, Y.M., Park, H.J., Yim, S.D., Baek, N.I., Lee, C.H., An, G. and Woo, Y.M., 2006, Transgenic rice lines expressing maize C1 and R-S regulatory genes produce various flavonoids in the endosperm, Plant Biotechnol. J. 4: 303-315.
Shih, C.H., Chu, H., Tang, L.K., Sakamoto, W., Maekawa, M., Chu, I.K. and Lo, C., 2008, Functional characterization of key structural genes in rice flavonoid biosynthesis, Planta 228: 1043-1054.
Sun, X., Zhang, Z., Chen, C., Wu, W., Ren, N., Jiang, C. and Zhang, H., 2018, The C-S-A gene system regulates hull pigmentation and reveals evolution of anthocyanin biosynthesis pathway in rice, J. Exp. Bot. 69: 1485-1498.
Toki, S., 1997, Rapid and efficient Agrobacte rium-mediated transformation in rice, Plant Mol. Biol. Rep. 15: 16-21.
Zhu, Q., Yu, S., Zeng, D., Liu, H., Wang, H., Yang, Z. and Zhao, X., 2017, Development of “Purple Endosperm Rice” by engineering anthocyanin biosynthesis in the endosperm with a high-efficiency transgene stacking system, Mol. Plant 10: 918-929.