The leucoanthocyantin reducase gene (LAR), which was an important functional gene of catechins biosynthesis pathway, was cloned from tea plant by EST sequencing and rapid amplification of cDNA ends (RACE). The full-length cDNA of LAR is 1 301 bp (GenBank accession No. EF205148), containing a 1 029 bp ORF encoding a 342 amino acids protein, and its 3′ untranslated region has an obvious polyadenylation signal. The deduced protein molecular weight was 37.5 kD and its theoretical isoelectric point was 5.81. The gene was then constructed into expression vector pET-32a(+) for over expression in prokaryotic cells. The SDS-PAGE showed that induced by IPTG, the leucoanthocyantin reducase proteins was expressed in Escherichia coli BL21, and its molecular weigh was found to be about 60 kD by checking with SDS-PAGE, nearly equal to the predicted. The deduced amino acid sequence of LAR from tea plant showed high identity with that of other plants, for instance 71%, 70% and 68% with Vitis vinifera, Gossypium arboretum and Fragaria ananassa, respectively. Four different catechin content cultivars were selected from our tea germplasm appraisal database to assay the gene expression level of the seven genes, flavonol synthase, dihydroflavonol-4-reductase, leucoanthocyanidin reductase, anthocyanidin synthase, etc, which were involved in the flavonoids biosynthesis. The result showed that, the DFR and LAR transcripts were expressed increased with the increasing of tea catechin contents. Nevertheless, the others did not show this tendency clearly.
MA Chun-lei
,
QIAO Xiao-yan
,
CHEN Liang
. Cloning and Expression Analysis of Leucoanthocyantin Reducase Gene of Tea Plant (Camellia sinensis)[J]. Journal of Tea Science, 2010
, 30(1)
: 27
-36
.
DOI: 10.13305/j.cnki.jts.2010.01.005
[1] Lin YL, Juan IM, Chen YL, et al. Composition of polyphenols in fresh tea leaves and associations of their oxygen-radicalabsorbing capacity with antiproliferative actions in fibroblast cells[J]. Journal of Agricultural and Food Chemistry, 1996, 44: 1387-1394.
[2] Wang ZY, Cheng SJ, Zhou ZC, et al. Antimutagenic activity of green tea polyphenols[J]. Mutation Research, 1989, 223: 273-285.
[3] Devic M, Guilleminot J, Debeaujon I, et al. The BANYULS gene encodes a DFR-like protein and is a marker of early seed coat development[J]. The Plant Journal, 1999, 19(4): 387-398.
[4] Marles MA, Ray H, Gruber MY.New perspectives on proanthocyanidin biochemistry and molecular regulation[J]. Phytochemistry, 2003, 64(2): 367-383.
[5] Dixon RA, Xie DY, Sharma SB.Proanthocyanidins a final frontier in flavonoid research?[J]. New Phytologist, 2005, 165(1): 9-28.
[6] Xie DY, Sharma SB, Paiva NL, et al. Role of anthocyanidin reductase, encoded by BANYULS in plant flavonoid biosynthesis[J]. Science, 2003, 299(5605):396-399.
[7] 明亚玲, 骆丹, 徐晶, 等. 茶多酚单体和黄芩苷对紫外线辐射皮肤成纤维细胞的影响[J]. 中国美容医学, 2005, 14(5): 541-544.
[8] 宋秀祖, 夏济平, 毕志刚. 茶多酚单体对中波紫外线辐射培养的成纤维细胞氧化损伤的保护机制研究[J]. 临床皮肤科杂志, 2003, 32(6): 315-317.
[9] Yu HN, Yin JJ, Shen SR.Growth inhibition of prostate cancer cells by epigallocatechin gallate in the presence of Cu2+[J]. Journal of Agricultural and Food Chemistry, 2004, 52(3): 4622-4661.
[10] Dixon RA, Steele CL.Flavonoids and isoflavonoids-a gold mine for metabolic engineering[J]. Trends Plant Science, 1999, 4(10): 394-400.
[11] Choi SU, Ryu SY, Yoon SK, et al. Effects of flavonoids on the growth and cell cycle of cancer cells[J]. Anticancer Research, 1999, 19(6B): 5229-5233.
[12] 马春雷, 陈亮. 茶树功能基因分离克隆研究进展[J]. 分子植物育种, 2006, 4(3S): 16-22.
[13] 陆建良, 林晨, 骆颖颖, 等. 茶树重要功能基因克隆研究进展[J]. 茶叶科学, 2007, 27(2): 95-103.
[14] Chen L, Zhao LP, Gao QK.Generation and analysis of expressed sequence tags from the tender shoot cDNA library of tea plant (Camellia sinensis)[J]. Plant Science, 2005, 168:359-363.
[15] 赵丽萍, 高其康, 陈亮, 等. 茶树基因芯片的研制和初步应用[J]. 茶叶科学, 2006, 26(3): 166-170.
[16] 张亚丽, 赵丽萍, 马春雷, 等. 茶树亲环素基因cDNA 全长的分析鉴定与原核表达[J]. 茶叶科学, 2007, 27(2): 120-126.
[17] Thompson JD, Higgins DG, Gibson TJ.CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice[J]. Nucleic Acids Research, 1994, 22(22): 4673-4680.
[18] Felsenstein J.Confidence limits on phylogenies: an approach using the bootstrap[J]. Evolution, 1985, 39(4): 783-791.
[19] Saitou N, Nei M.The neighbor-joining method: a new method for reconstructing phylogenetic trees[J]. Molecular Biology and Evolution, 1987, 4(4): 406-425.
[20] Kumar S, Tamura K, Nei M.MEGA3: Integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment[J]. Briefings in Bioinformatics, 2004, 5(2): 150-163.
[21] Furukawa T, Eshima A, Kouya M, et al. Coordinate expression of genes involved in catechin biosynthesis in Polygonum hydropiper cells[J]. Plant Cell Reports, 2002, 21(4): 385-389.
[22] Punyasiri PA, Abeysinghe IS, Kumar V, et al. Flavonoid biosynthesis in the tea plant Camellia sinensis:Properties of enzymes of the prominent epicatechin and catechin pathway[J]. Archives of Biochemistry and Biophysics, 2004(431): 22-30.
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