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Int. J. Mol. Sci. 2016, 17(2), 261; doi:10.3390/ijms17020261

Cloning and Characterization of a Flavonoid 3′-Hydroxylase Gene from Tea Plant (Camellia sinensis)

1
Horticulture, Northwest A&F University, 3 Taicheng Road, Yangling 712100, China
2
Conagen Inc., 15 DeAngelo Dr., Bedford, MA 01730, USA
3
Tea Research Institute, Chinese Academy of Agricultural Sciences, 9 Meiling South Road, Hangzhou 310008, China
*
Authors to whom correspondence should be addressed.
Academic Editor: Marcello Iriti
Received: 9 December 2015 / Revised: 3 February 2016 / Accepted: 15 February 2016 / Published: 22 February 2016
(This article belongs to the Special Issue Molecular Research in Plant Secondary Metabolism 2015)
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Abstract

Tea leaves contain abundant flavan-3-ols, which include dihydroxylated and trihydroxylated catechins. Flavonoid 3′-hydroxylase (F3′H: EC 1.14.13.21) is one of the enzymes in the establishment of the hydroxylation pattern. A gene encoding F3′H, designated as CsF3H, was isolated from Camellia sinensis with a homology-based cloning technique and deposited in the GenBank (GenBank ID: KT180309). Bioinformatic analysis revealed that CsF3H was highly homologous with the characterized F3Hs from other plant species. Four conserved cytochrome P450-featured motifs and three F3′H-specific conserved motifs were discovered in the protein sequence of CsF3H. Enzymatic analysis of the heterologously expressed CsF3H in yeast demonstrated that tea F3′H catalyzed the 3′-hydroxylation of naringenin, dihydrokaempferol and kaempferol. Apparent Km values for these substrates were 17.08, 143.64 and 68.06 μM, and their apparent Vmax values were 0.98, 0.19 and 0.44 pM·min−1, respectively. Transcription level of CsF3H in the new shoots, during tea seed germination was measured, along with that of other key genes for flavonoid biosynthesis using real-time PCR technique. The changes in 3′,4′-flavan-3-ols, 3′,4′,5′-flavan-3-ols and flavan-3-ols, were consistent with the expression level of CsF3H and other related genes in the leaves. In the study of nitrogen supply for the tea plant growth, our results showed the expression level of CsF3H and all other tested genes increased in response to nitrogen depletion after 12 days of treatment, in agreement with a corresponding increase in 3′,4′-catechins, 3′,4′,5′-catechins and flavan 3-ols content in the leaves. All these results suggest the importance of CsF3H in the biosynthesis of 3′,4′-catechins, 3′,4′,5′-catechins and flavan 3-ols in tea leaves. View Full-Text
Keywords: flavonoid 3′-hydroxylase; heterologous expression; flavan 3-ols; nitrogen depletion; Camellia sinensis flavonoid 3′-hydroxylase; heterologous expression; flavan 3-ols; nitrogen depletion; Camellia sinensis
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MDPI and ACS Style

Zhou, T.-S.; Zhou, R.; Yu, Y.-B.; Xiao, Y.; Li, D.-H.; Xiao, B.; Yu, O.; Yang, Y.-J. Cloning and Characterization of a Flavonoid 3′-Hydroxylase Gene from Tea Plant (Camellia sinensis). Int. J. Mol. Sci. 2016, 17, 261.

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