紫花苜蓿二氢黄酮醇还原酶基因(MsDFR)的克隆与分析

摘要/Abstract

摘要： 二氢黄酮醇还原酶(dihydroflavonol reductase,DFR)是缩合单宁生物合成途径中的关键酶,在单宁的合成中起着重要的作用。根据同源克隆的原理,利用RACE技术,从“中苜一号”苜蓿中克隆得到DFR基因(MsDFR),并对其进行了序列分析及不同胁迫条件下的表达模式分析。结果表明,MsDFR基因cDNA全长1 402 bp,包括开放阅读框1 023 bp,编码340个氨基酸,在N端存在1个NADP结合位点“VTGASGFIGSWLVMRLMERGY”,中部存在1个底物特异性结合的氨基酸基序“TLNVTEDQKPLWDESCWSDVEFCRRV”。实时荧光定量PCR结果表明,该基因在荚果中表达量较高,根中较弱;在NaCl和GA₃诱导下,MsDFR基因表达受到抑制;在黑暗条件下,该基因被诱导表达。由此推测“中苜一号”苜蓿中可能存在不依赖于GA₃信号的单宁合成途径。

Abstract: Dihydroflavonol reductase (DFR) is a key enzyme in condensed tannin synthesis pathway. A cDNA of DFR gene is cloned from alfalfa (Medicago sativa) by using rapid amplification of cDNA ends (RACE) method. The expression pattern of MsDFR under different stresses was analyzed by qRT-PCR. The bio-informatical analysis showed that the full-length of cDNA sequence is 1 402 bp and includes a 1 023 bp open reading frame which encodes a 340-amino-acid polypeptide. A nicotinamide adenine dinucleotide phosphate (NADP) binding site “VTGASGFIGSWLVMRLMERGY” and a substrate specificity motif “TLNVTEDQKPLWDESCWSDVEFCRRV” were detected in the deduced amino acid sequence of MsDFR. The results of Real-time PCR indicate that the expression level of MsDFR gene is highest in pods, and least in root. The expression of MsDFR gene under stress of NaCl and GA₃ is down-regulated. In dark environment, the MsDFR gene expression was induced. There is a gibberellin (GA) signaling-independent pathway of tannin synthesis in alfalfa.

中图分类号:

董洁,王学敏,王赞,高洪文,孙桂枝. 紫花苜蓿二氢黄酮醇还原酶基因(MsDFR)的克隆与分析[J]. 草业学报, 2012, 21(2): 123-132.

DONG Jie, WANG Xue-min, WANG Zan, GAO Hong-wen, SUN Gui-zhi. Cloning and analysis of dihydroflavonol reductase (DFR) gene from Medicago sativa[J]. Acta Prataculturae Sinica, 2012, 21(2): 123-132.

参考文献

[1] 韩明鹏, 王颜华, 高永革, 等. 高温胁迫下紫花苜蓿抑制消减文库的构建[J]. 草业学报, 2011, 20(5): 126-132.
[2] 姜健, 杨宝灵, 夏彤, 等. 紫花苜蓿耐盐种植资源的遗传多样性分析[J]. 草业学报, 2011, 20(5): 119-125.
[3] Clark R T, Reid C S. Foamy bloat of cattle. A review[J]. Dairy Science, 1974, 57: 753-785.
[4] 戎郁萍, 王堃. 苜蓿与放牧家畜臌涨病研究概况[J]. 草业科学, 2004, 21(12): 103-107.
[5] Pierre P, Thierry G, Béatrice L E, et al. Crystal structure of grape dihydroflavonol 4-Reductase, a key enzyme in flavonoid Biosynthesis[J]. Journal of Molecular Biology, 2007, 368: 1345-1357.
[6] 刘娟, 冯群芳, 张杰. 二氢黄酮醇4-还原酶基因(DFR)与花色的修饰[J]. 植物生理学通讯, 2005, 41(6): 715-716.
[7] 宋艳波, 吴国良, 牛洪斌. 改良CTAB法在核桃叶片基因组DNA提取中的应用研究[J]. 山西农业大学学报(自然科学版), 2011, (2): 109-112.
[8] Kenneth J, Thomas D. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCT method[J]. Methods, 2001, 25(4): 402-408.
[9] Baker M E, Blasco R. Expansion of the mammalian 3β-hydroxysteroid dehydrogenase/plant dihydroflavonol reductase superfamily to include a bacterial cholesterol dehydrogenase, a bacterial UDP-galactose-4-epimerase, and open reading frames in vaccinia virus and fish lymphocystis disease virus[J]. Federation of European Biochemical Societies Letters, 1992, 301: 89-93.
[10] Deborah G, Jan V D, Nabila Y, et al. A novel aromatic alcohol dehydrogenase in higher plants: molecular cloning and expression[J]. Plant Molecular Biology, 1998, 36: 755-765.
[11] Jordi B, Sílvia A, Roser G D, et al. The refined crystal structure of Drosophila lebanonensis alcohol dehydrogenase at 1.9  resolution[J]. Journal of Molecular Biology, 1998, 282(2): 383-399.
[12] Benach J, Filling C, Oppermann C T, et al. Structure of bacterial 3β/17β-hydroxysteroid dehydrogenase at 1.2  resolution: a model for multiple steroid recognition[J]. Biochemistry, 2002, 41(50): 14659-14668.
[13] Johnson E T, Ryu S, Yi H, et al. Alteration of a single amino acid changes the substrate specificity of dihydrofavonol 4-reductase[J]. The Plant Journal, 2001, 25(3): 325-333.
[14] Robbins M P, Bavage A D, Allison G, et al. A comparison of two strategies to modify the hydroxylation of condensed tannin polymers in Lotus corniculatus L.[J]. Phytochemistry, 2005, 66: 991-999.
[15] Forkmann G. Flavonoids as flower pigments: the formation of the natural spectrum and its extension by genetic engineering[J]. Plant Breeding, 1991, 106(1): 1-26.
[16] Forkmann G, Stefan M. Metabolic engineering and applications of flavonoids[J]. Current Opinion Biotechnology, 2001, 2(2): 155-160.
[17] Charrier B, Coronado C, Kondorosi A, et al. Molecular characterization and expression of alfalfa (Medicago sativa L.) flavanone-3-hydroxylase and dihydroflavonol-4-reductase encoding genes[J]. Plant Molecular Biology, 1995, 29: 773-786.
[18] Johnson E T, Yi H, Shin B, et al. Cymbidium hybrida dihydroflavonol 4-reductase does not efficiently reduce dihydrokaempferol to produce orange pelargonidin- type anthocyanins[J]. The Plant Journal, 1999, 19(3): 81-85.
[19] Thoden J B, Frey P A,Holden H M. Crystal structures of the oxidized and reduced forms of UDP-galactose 4-epimerase isolated from Escherichia coil[J]. Biochemistry, 1996, 35(8): 2557-2566.
[20] Thoden J B, Frey P A, Holden H M. High-resolution X-ray structure of UDP-galactose 4-epimerase complexed with UDP-phenol[J]. Protein Science, 1996, 5(11): 2149-2161.
[21] Thoden J B, Hegeman A D, Wesenberg G, et al. Structure analysis of UDP-sugar binding to UDP-galactose 4-epimerase from Escherichia coli[J]. Biochemistry, 1997, 36(21): 6294-6304.
[22] Inagaki Y, Johzuka H Y, Mori T, et al. Genomic organization of the genes encoding dihydroflavonol 4-reductase for flower pigmentation in the Japanese and common morning glories[J]. Gene, 1999, 226(2): 181-188.
[23] Tunen A J, Koes R E, Spelt C E, et al. Cloning of the two chalcone flavanone isomerase genes from Petunia hybrida: coordinate, light-regulated and differential expression of flavonoid genes[J]. The European Molecular Biology Organization Journal, 1988, 7(5): 1257-1263.
[24] Helariutta Y, Elomaa P, Kotilainen M, et al. Cloning of cDNA coding for dihydroflavonol-4-reductase (DFR) and characterization of dfr expression in the corollas of Gerbera hybrida var. Regina (Compositae)[J]. Plant Molecular Biology, 1993, 22(2): 183-193.
[25] Helariutta Y, Kotilainen M, Elomaa P, et al. Duplication and functional divergence in the chalcone synthase gene family of Asteraceae: evolution with substrate change and catalytic simplification[J]. Proceedings of the National Academy of Sciences of the United States of America, 1996, 93(17): 9033-9038.
[26] Helariutta Y, Kotilainen M, Elomaa P, et al. Gerbera hybrida (Asteraceae) imposes regulation at several anatomical levels during inflorescence development on the gene for dihydroflavonol-4-reductase[J]. Plant Molecular Biology, 1995, 28(5): 935-941.
[27] Helariutta Y, Elomaa P, Kotilainen M, et al. Chalcone synthase-like genes active during corolla development are differentially expressed and encode enzymes with different catalytic properties in Gerbera hybrida (Asteraceae)[J]. Plant Molecular Biology, 1995, 8(1): 47-60.
[28] 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 Physiology, 2004, 134(3): 979-994.
[29] Singh K, Kumar S, Yadav S K, et al. Characterization of dihydroflavonol 4-reductase cDNA in tea [Camellia sinensis(L.) O. Kuntze][J]. Plant Biotechnology Reports, 2009, 3: 95-101.
[30] Jeyaramraja P R, Pius P K, Kumar R R, et al. Soil moisyure stress-induced alteration in bioconstituents determining tea quality[J]. Journal of the Science of Food and Agriculture, 2003, 230: 1187-1191.
[31] 廖祥儒, 张蕾, 徐景智, 等. 光在植物生长发育中的作用[J]. 河北大学学报(自然科学版), 2001, 21(3): 341-346.
[32] 李平, 杨玲玲, 陈其新, 等. 两种策略分别克隆紫花苜蓿光敏色素A、B基因[J]. 草业学报, 2011, 20(6): 85-92.
[33] 赵德修, 李茂寅, 邢建民, 等. 光质、光强和光期对水母莲愈伤组织生长和黄酮生物合成的影响[J]. 植物生理学报, 1999, 25(2): 27-132.
[34] 王曼, 王小菁. 蓝光、紫外光的受体及其对CHS表达诱导的研究[J]. 植物学通报, 2002, 19(3): 265-271.
[35] 王曼, 王小菁. 蓝光和蔗糖对拟南芥花色素苷积累和CHS基因表达的影响[J]. 热带亚热带植物学报, 2004, 12(3): 252-256.
[36] Rabino I, Mancinelli A L. Light, temperature, and anthocyanin produetion[J]. Plant Physiology, 1986, 81(3): 922-924.
[37] Dong Y H, Beuning L, Davies K, et al. Expression of pigmentation genes and Photo-regulation of anthocyanin biosynthesis in developing Royal Gala apple flowers[J]. Australian Journal of Plant Physiology, 1998, 25(2): 245-252.
[38] 文樵夫, 沈红香, 姚允聪, 等. 苹果属观赏海棠McDFR的克隆及不同叶色品种间的表达差异[J]. 林业科学, 2010, 46(11): 16-24.
[39] Joseph M, Erich G, Ronald K. How genes paint flowers and seeds[J]. Trends Plant Science, 1998, 3(6): 212-218.
[40] Shirley B W, Hanley S, Goodman H M. Effects of ionizing radiation on a plant genome: analysis of two Arabidopsis transparent testa mutations[J]. Plant Cell, 1992, 4(3): 333-347.
[41] Katz A, Weiss D. Photocontrol of chs gene expression in petunia flowers[J]. Physiologia Plantarum, 1998, 102(2): 210-216.
[42] 张艳, 柴岩, 冯佰利, 等. 苦荞和甜荞查尔酮合成酶基因的克隆及序列分析[J]. 西北植物学报, 2008, 28(3): 447-45.
[43] 徐纪尊, 王丽辉, 潘庆玉. 观赏植物花色基因转化的研究进展[J]. 中国农业科技导报, 2006, 8(5): 56-60.
[44] Kaneko M, Itoh H, Inukai Y, et al. Where to gibberellin biosynthesis and gibberellin signaling occur in rice plants[J]. The Plant Journal, 2003, 35(1): 104-115.
[45] Stoddart J L, Thomas H, Grierson D. Genetic and hormonal regulation of stature[A]. Developmental Mutants in Plants[M]. Cambridge University Press, 1987: 155-180.
[46] 孟祥春, 彭建宗, 王小菁. 光和糖对非洲菊花色素苷积累及CHS、DFR基因表达的影响[J]. 园艺学报, 2007, 34(1): 227-230.
[47] Hosokawa K, Fukunaga Y, Fukushi E, et al. Production of acylated anthocyanins by blue flowers of Hyacinthus orient alis regenerated in vitro[J]. Phytochemistry, 1996, 41(6): 1531-1533.
[48] Silverstone A L, Chang C, Krol E, et al. Developmental regulation of the gibberellin biosynthetic gene GA1 in Arabidopsis thaliana[J]. The Plant Journal, 1997, 12(1): 9-19.