茶树花青素还原酶是催化非酯型儿茶素EC和EGC合成的关键酶。采用RT-PCR技术,获得了茶树花青素还原酶基因的开放阅读框,它编码含337个氨基酸的蛋白质,推测分子量为37kD,等电点为6.54;成功地将该基因重组到表达载体pET32a(+)上,并在大肠杆菌rosetta中进行原核表达;优化了原核表达中诱导时间、诱导温度、IPTG浓度、氨苄青霉素浓度,纯化出目的蛋白。HPLC检测表明,目的蛋白具有ANR酶活性。
骆洋
,
王弘雪
,
王云生
,
卢忠尉
,
刘亚军
,
单育
,
陈啸天
,
叶辉
,
高丽萍
,
夏涛
. 茶树花青素还原酶基因在大肠杆菌中的表达及优化[J]. 茶叶科学, 2011
, 31(4)
: 326
-332
.
DOI: 10.13305/j.cnki.jts.2011.04.014
Anthocyanin reductase is a key enzyme in the biosynthesis of EC and EGC in tea plant. The open reading frame of anthocyanin reductase gene (ANR), which encoding a 337 amino acids protein, was cloned from tea plant 〔Camellia sinensis (L.) O. Kuntze 〕 by RT-PCR. The deduced protein molecular weight was 37kD and its theoretical isoelectric point was 6.54. The gene was cloned into the expression vector pET32a (+) for expression in prokaryotic cells. The SDS-PAGE results showed that the anthocyanin reductase peoteins was expressed in Escherichia coli rosetta. The optimal inducing conditions including time, temperature, IPTG concentration, ampicillin concentration were studied. The deduced protein was purified and its activity was detected by HPLC.
[1] 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.
[2] Dalluge J J, Nelson B C.Determination of tea catechins[J]. J Chromatogr A, 2000, 881(1/2): 411-424.
[3] Winkel-Shirley B.Flavonoid biosynthesis. A colorful model for genetics, biochemistry, cell biology, and biotechnology[J]. Plant Physiol, 2001, 126(2): 485-493.
[4] 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.
[5] Xie D Y, Sharma S B, Wright E, et al. Metabolic engineering of proanthocyanidins through co-expression of anthocyanidin reductase and the PAP1 MYB transcription factor[J]. Plant J, 2006, 45(6): 895-907.
[6] Xie D Y, Sharma S B, Dixon R A.Anthocyanidin reductases from Medicago truncatula and Arabidopsis thaliana[J]. Arch Biochem Biophys, 2004, 422(1): 91-102.
[7] Xie D Y, Jackson LA, Cooper JD, 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.
[8] Grotewold E.The Science of Flavonoids[M]. New York: Springer, 2006: 75-95.
[9] Stafford H A.In Flavonoid Metabolism[M]. New York: CRC Press, 1990: 63-99.
[10] Stafford H A, Lester H H.Flavan-3-ol Biosynthesis: The Conversion of (+)-Dihydroquercetin and Flavan-3,4-cis-Diol (Leucocyanidin) to (+)-Catechin by Reductases Extracted from Cell Suspension Cultures of Douglas Fir[J]. Plant Physiol, 1984, 76(1): 184-186.
[11] Stafford H A, Lester H H.Flavan-3-ol Biosynthesis: The Conversion of (+)-Dihydromyricetin to Its Flavan-3,4-Diol (Leucodelphinidin) and to (+)-Gallocatechin by Reductases Extracted from Tissue Cultures of Ginkgo biloba and Pseudotsuga menziesii[J]. Plant Physiol, 1985, 78(4): 791-794.
[12] Punyasiri P A, Abeysinghe I S, Kumar V, et al. Flavonoid biosynthesis in the tea plant Camellia sinensis: properties of enzymes of the prominent epicatechin and catechin pathways[J]. Arch Biochem Biophys, 2004, 431(1): 22-30.
[13] Singh K, Rani A, Paul A, et al. Differential display mediated cloning of anthocyanidin reductase gene from tea (Camellia sinensis) and its relationship with the concentration of epicatechins[J]. Tree Physiol, 2009, 29(6): 837-846.
[14] 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.
[15] 江昌俊, 王朝霞, 李叶云. 茶树中提纯总RNA的研究[J]. 茶叶科学, 2000, 20(1): 27-29.
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