An overexpression of chalcone reductase of Pueraria montana var. lobata alters biosynthesis of anthocyanin and 5′-deoxyflavonoids in transgenic tobacco☆

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Abstract

We isolated the chalcone reductase (pl-chr) gene of Pueraria montana var. lobata by using a PCR strategy from cDNA pools of storage roots. A high level of expression of RNA was found in both stems and roots. The genomic Southern blot result suggests that pl-chr exists as a member of a small gene family. By introducing a pl-chr gene under the control of the 35S CaMV promoter into the pink-flowering Xanthi line of Nicotiana tabacum, the flower color was changed from pink to white-to-pink. The contents of anthocyanin in the flowers of the transgenic lines were dramatically decreased by 40%, but the total UV absorption compounds remained unchanged. The production of liquiritigenin in pl-chr overexpressed transgenic tobacco lines was confirmed by HPLC and MS analysis. The introduction of pl-chr gene provides a method to redirect the flavonoid pathway into 5′-deoxyflavonoid production in non-legume crops, in order to manipulate the phenylpropanoid pathway for isoflavonoid production.

Section snippets

Materials and methods

Cloning of chalcone reductase cDNA. We used a polymerase chain reaction (PCR) strategy to clone P. montana var. lobata chalcone reductase cDNA. The storage roots of P. montana var. lobata grown in a field were used for extraction of total RNA. Total RNA was isolated from the storage root according to the phenol/SDS methods [19], and first-strand cDNA was synthesized with oligo(dT)-anchor primer using the 5′/3′-rapid amplification of a cDNA ends (RACE) Kit (Roche Molecular Biochemicals). Based

Cloning and characterization of pl-chr

The cDNA sequence is 1109 nucleotides long and consists of an open reading frame encoding a peptide of 314 amino acids (Fig. 2), with a calculated molecular mass of 35.4 kDa and an isoelectric point of 6.4. The BLAST analysis [24] on the homology of the deduced amino acid sequence shows high homologies with soybean (X55730), G. echinata (D83718), and M. sativa (U13925) (93%, 87%, and 83% identities, respectively) (Fig. 2). Moreover, pl-chr, like other aldo/keto reductases, contains a number of K+

Acknowledgements

This research was supported by a Grant (PF003102-03) from Plant Diversity Research Center of 21st Century Frontier Research Program funded by Ministry of Science and Technology of Korean government.

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    ☆

    Pueraria montana var. lobata chalcone reductase sequence has been submitted to GenBank and the Accession No. awarded is AF462632.

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