Clone, Expression and Functional Analysis of Dihydroflavonol 4-Reductase Gene of Tea Plant (Camellia sinensis)

WANG Yun-sheng; XU Yu-jiao; HU Xiao-jing; JIANG Xiao-lan; YANG Qing; LI Wei-wei; LIU Ya-jun; GAO Li-ping; XIA Tao

doi:10.13305/j.cnki.jts.2013.03.010

PDF(1272 KB)

Journal of Tea Science ›› 2013, Vol. 33 ›› Issue (3) : 193-201. DOI: 10.13305/j.cnki.jts.2013.03.010

Clone, Expression and Functional Analysis of Dihydroflavonol 4-Reductase Gene of Tea Plant (Camellia sinensis)

WANG Yun-sheng^1,2, XU Yu-jiao¹, HU Xiao-jing¹, JIANG Xiao-lan², YANG Qing¹, LI Wei-wei¹, LIU Ya-jun¹, GAO Li-ping^1,*, XIA Tao^2,*

Author information +

History +

Abstract

Dihydroflavonol 4-reductase (DFR) is a key enzyme in the biosynthesis of catechins in tea plant. However, the functions and the zymologic properties of DFR were not deeply identified in recent researches. The open reading frame of DFR gene, which encoding a 347 amino acids protein, was cloned from tea plant (Camellia sinensis) by RT-PCR. The deduced protein molecular weight was 38.69βkD and its theoretical isoelectric point was 6.02. The gene was cloned into the expression vector SUMO for expression in prokaryotic cells. The SDS-PAGE results showed that the dihydroflavonol 4-reductase peoteins was expressed in Escherichia coli BL21. The optimal inducing conditions including time, temperature and IPTG concentration were studied. The deduced protein was purified and its activity was detected by HPLC-MS method. The results indicated that purified protein showed the DFR activity, catalyzed the reduction reaction of DHQ and DHM. The research provides a valuable foundation for better understanding the substrate specificity and enzymatic properties of CsDFR.

Key words

dihydroflavonol 4-reductase / enzyme activity / prokaryotic expression / tea plant (Camellia sinensis)

Cite this article

EndNote

Ris (Procite)

Bibtex

Download Citations

WANG Yun-sheng, XU Yu-jiao, HU Xiao-jing, JIANG Xiao-lan, YANG Qing, LI Wei-wei, LIU Ya-jun, GAO Li-ping, XIA Tao. Clone, Expression and Functional Analysis of Dihydroflavonol 4-Reductase Gene of Tea Plant (Camellia sinensis)[J]. Journal of Tea Science. 2013, 33(3): 193-201 https://doi.org/10.13305/j.cnki.jts.2013.03.010

References

[1] Park J S, Kim J B, Hahn B S, et al. EST analysis of genes involved in secondary metabolism in Camellia sinensis (tea), using suppression subtractive hybridization[J]. Plant Science, 2004, 166(4): 953-961.
[2] Wan X, Li D, Zhang Z.Antioxidant properties and mechanisms of tea ployphenols. In: Ho C T, Lin J K, Shahidi F, eds. Tea and Tea Products[M]. New York: CRC press, 2008: 131-159.
[3] Dalluge J J, Nelson B C.Determination of tea catechins[J]. J Chromatogr A, 2000, 881(1): 411-424.
[4] 夏涛, 高丽萍. 类黄酮及茶儿茶素生物合成途径及其调控研究进展[J]. 中国农业科学, 2009, 42(8): 2899-2908.
[5] Pang Y, Peel G J, Wright E, et al. Early steps in proanthocyanidin biosynthesis in the model legume Medicago truncatula[J]. Plant Physiol, 2007, 145(3): 601-615.
[6] Winkel-Shirley B.The biosynthesis of flavonoids. In: Grotewold E, Ed. The Science of Flavonoids[M]. New York: Springer Science & Business Media, 2006: 75-95.
[7] Boase M R, Lewis D H, Davies K M, et al. Isolation and antisense suppression of flavonoid 3', 5'-hydroxylase modifies flower pigments and colour in cyclamen[J]. BMC Plant Biol, 2010, 10: 107.
[8] Han Y, Vimolmangkang S, Soria-Guerra R E, et al. Ectopic expression of apple F3'H genes contributes to anthocyanin accumulation in the Arabidopsis tt7 mutant grown under nitrogen stress[J]. Plant Physiol, 2010, 153(2): 806-820.
[9] Punyasiri P A N, Abeysinghe I S B, Kumar V, et al. Flavonoid biosynthesis in the tea plant Camellia sinensis: properties of enzymes of the prominent epicatechin and catechin pathways[J]. Archives of Biochemistry and Biophysics, 2004, 431(1): 22-30.
[10] Xie D Y, Sharma S B, Paiva N L, et al. Role of anthocyanidin reductase, encoded by BANYULS in plant flavonoid biosynthesis[J]. Science, 2003, 299(5605): 396-399.
[11] 马春雷, 乔小燕, 陈亮. 茶树无色花色素还原酶基因克隆及表达分析[J]. 茶叶科学, 2010, 30(1): 27-36.
[12] Eungwanichayapant P D, Popluechai S.Accumulation of catechins in tea in relation to accumulation of mRNA from genes involved in catechin biosynthesis[J]. Plant Physiol Biochem, 2009, 47(2): 94-97.
[13] Mamati G E, Liang Y, Lu J.Expression of basic genes involved in tea polyphenol synthesis in relation to accumulation of catechins and total tea polyphenols[J]. Journal of the Science of Food and Agriculture, 2006, 86(3): 459-464.
[14] 江昌俊, 王朝霞. 茶树中提纯总RNA的研究[J]. 茶叶科学, 2000, 20(1): 27-29.
[15] 张宪林, 高丽萍, 夏涛, 等. 茶树新梢中非酯型儿茶素及其合成酶的变化规律[J]. 茶叶科学, 2009, 29(5): 365-371.
[16] Beld M, Martin C, Huits H, et al. Flavonoid synthesis in Petunia hybrida: partial characterization of dihydroflavonol-4-reductase genes[J]. Plant Mol Biol, 1989, 13(5): 491-502.
[17] Johnson E T, Ryu S, Yi H, et al. Alteration of a single amino acid changes the substrate specificity of dihydroflavonol 4-reductase[J]. Plant J, 2001, 25(3): 325-333.
[18] Wang Y S, Gao L P, Wang Z R, et al. The influence of light on the biosynthesis of catechins in tea [Camellia sinensis (L.) O. Kuntze][J]. Scientia Horticulturae, 2012, 133: 72-83.
[19] Shimada N, Sasaki R, Sato S, et al. A comprehensive analysis of six dihydroflavonol 4-reductases encoded by a gene cluster of the Lotus japonicus genome[J]. Journal of experimental botany, 2005, 56(419): 2573-2585.
[20] Xie D Y, Jackson L A, Cooper J D, et al. Molecular and biochemical analysis of two cDNA clones encoding dihydroflavonol-4-reductase from Medicago truncatula[J]. Plant Physiol, 2004, 134(3): 979-994.
[21] Petit P, Granier T, Estaintot B L, et al. Crystal structure of grape dihydroflavonol 4-reductase, a key enzyme in flavonoid biosynthesis[J]. J Mol Biol, 2007, 368(5): 1345-1357.