Cloning and Characterization of Chalcone Synthase Gene from Pueraria Candollei Var

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[P-P&F.20] Cloning and Characterization of Chalcone Synthase Gene from Pueraria candollei var. mirifica Piyachat Wiriyaampaiwong* 1, Pornthap Thanonkeo 1, Sudarat Thanonkeo 2 1Khon Kaen University, Thailand, 2Mahasarakham University, Thailand Pueraria candollei var. mirifica is an indigenous herb found in the North, West and Northeastern part of Thailand. This plant synthesized and accumulated several biological active compounds namely phytoestrogen such as miroestrol, deoxymiroestrol, daidzin, puerarin, genistin, mirificin, kawakhurin, coumestrol, daidzein and genistein. Chalcone synthase (CHS; EC 2.3.1.74), which catalyzes the formation of chalcone from p-coumaroyl and malonyl CoAs, is the key enzyme in flavonoid biosynthesis in plant. In this study, cloning and characterization of chalcone synthase gene ( CHS ) from P. candollei were carried out. Using a reverse transcription-polymerase chain reaction (RT-PCR) strategy, the CHS gene was cloned from P. candollei . The complete PcCHS coding sequence contained 1170 bp, encoded for a polypeptide of 389 amino acid residues with a calculated molecular mass of 42.8 kDa and pI of 5.31. Southern blot hybridization of genomic DNA fragments revealed the presence of multiple CHS genes in the P. candollei genome. Homology analysis revealed that the deduced amino acid sequence of PcCHS had 86-93% identity, whereas the nucleotide sequence had 81-95% identity to CHS of other plants, such as P. montana , Glycine max , Phaseolus vulgaris , Vigna unguiculata , Pisum sativum and Medicago sativa . Expression analysis of the PcCHS gene in different plant tissues by RT-PCR revealed that this gene is expressed in a tissue-specific manner. The expression of the PcCHS gene was remarkably induced by high temperature, UV-B and wounding treatments, suggesting that the CHS gene product may plays a crucial role in protecting plant from environmental or external stresses. The results in this study provide useful information for further work on production of phytoestrogen, an active compound in this plant. Key Words : Pueraria candollei var. mirifica , Chalcone synthase, Flavonoid biosynthesis References Austin, M.B. and Noel, J.P. 2003. The chalcone synthase superfamily of type III polyketide synthases. Natural Product Report. 20:79–110. Cherdshewasart, W., Subtang, S. and Dahlan, W. 2007. Major isoflavonoid contents of the phytoestrogen rich-herb Pueraria mirifica in comparison with Pueraria lobata . Journal of Pharmaceutical and Biomedical Analysis. 43: 428–434. Pang, Y., Shen, G., Wu, W., Liu, X., Lin, J., Tan, F., Sun, X. and Tang, K. 2005. Characterization and expression of chalcone synthase gene from Ginkgo biloba . Plant Science. 168: 1525-1531. Keywords: Pueraria candollei var. mirifica, Chalcone synthase, Flavonoid biosynthesis .

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