茶树类黄酮3′-羟化酶基因的克隆与表达特性分析

摘要/Abstract

摘要： 类黄酮3′-羟化酶（Flavonoid 3′-hydroxylase, F3′H）是细胞色素P450酶家族（Cytochrome P450,CYP450）的单加氧酶,在植物次生代谢和逆境调控方面起着重要的作用。本实验以茶树品种龙井43作为试验材料,利用RT-PCR方法,从cDNA中克隆得到茶树中编码类黄酮3′-羟化酶基因,命名为CsF3′H1。序列分析显示,CsF3′H1基因开放阅读框为1β530βbp,编码509个氨基酸,含有相对保守的P450酶系结合域。进化树分析表明,CsF3′H1与CsF3′H3的进化树关系相近,与CsF3′H2的进化树关系较远。序列多重比对显示,CsF3′H1蛋白具有F3′H蛋白的特征基序,并与高粱、猕猴桃、杨树、拟南芥和葡萄同源蛋白一致性为66.48%。氨基酸组分、理化性质、亲水性/疏水性和无序化分析显示,CsF3′H1是亲水性蛋白,无序化特征不明显。利用实时荧光定量PCR对CsF3′H1基因在茶树不同组织和不同激素处理下的表达谱进行检测,结果显示：不同组织中,CsF3′H1基因的表达水平在第一叶中最高,之后随着叶片的成熟逐渐下降,CsF3′H1基因在老叶和根中几乎不表达,表达水平从高到低依次为第一叶>第二叶>第三叶>第四叶>嫩茎>根>老叶;在不同激素处理中,CsF3′H1在SA激素处理下的表达量最高,为对照的2.24倍,在MeJA处理下表达量最低,为对照的0.43倍。

关键词: 茶树, 类黄酮3′-羟化酶;, 进化树分析, 组织表达, 激素处理

Abstract: Flavonoid 3′-hydroxylase (F3′H) belongs to the family of cytochrome P450 monooxygenases (CYP450) and plays important roles in plant secondary metabolism and stress reponses. In this study, a gene encoding F3’H-like protein was cloned by RT-PCR method using a cDNA template from tea (Camellia sinensis) cultivar ‘Longjing43’. This gene is named as CsF3′H1. Sequence analysis showed that the open reading frame of CsF3’H1 was 1,530 bp length, encoding 509 amino acids. CsF3′H1 protein contains the conserved binding domain of P450 enzyme. Analysis of phylogenetic tree showed that CsF3'H1 protein has high similarity with CsF3'H3 protein and has low similarity with CsF3'H2 protein. Multiple alignments showed that CsF3'H1 protein has high similarity with the F3′H homologs from Sorghum bicolor, Actinidia chinensis, Populus trichocarpa, Arabidopsis thaliana, and Vitis vinifera (66.48% identity). CsF3'H1 protein contains the characterized motifs of F3'H-type protein. Analysis of amino acid composition, physical and chemical properties, hydrophilicity/hydrophobicity, and disordered residues of CsF3′H1 protein showed that the disordered residues of CsF3′H1 protein are not obvious and most amino acids of CsF3′H1 protein are hydrophilic. The expression profiles of CsF3′H1 gene in different tissues of tea plant or under hormonal treatments were detected using quantitative real-time PCR analysis. Results showed that: CsF3'H1 gene has the highest expression level in the first leaf. The tissue expression profiles showed that the successive order of CsF3'H1 gene expression levels was the first leaf > the second leaf > the third leaf > the fourth leaf > stem > roo > old leaf. The expression level of CsF3'H1 gene was highest under SA treatment, which was 2.24 times high than the control. CsF3'H1 gene had the lowest expression level under MeJA treatment.

Key words: Camellia sinensis, flavonoid 3′-hydroxylase, phylogenetic tree, tissue expression, hormonal treatments

中图分类号:

S571.1
Q52

王文丽, 吴致君, 刘志薇, 王永鑫, 李辉, 崔新, 庄静. 茶树类黄酮3′-羟化酶基因的克隆与表达特性分析[J]. 茶叶科学, 2017, 37(1): 108-118.

WANG Wenli, WU Zhijun, LIU Zhiwei, WANG Yongxin, LI Hui, CUI Xin, ZHUANG Jing. Cloning and Expression Analysis of the Gene Encoding Flavonoid 3′-Hydroxylase in Tea Plant (Camellia sinensis)[J]. Journal of Tea Science, 2017, 37(1): 108-118.

参考文献 31

[1]	侯杰, 佟玲, 崔国新, 等. 植物类黄酮3′羟化酶F3′H基因的研究进展[J]. 植物生理学报, 2011, 7(47): 641-647.
[2]	De Vetten N, Ter Horst J, Van Schaik HP, et al.A cytochrome b5 is required for full activity of flavonoid 3′, 5′-hydroxylase, a cytochrome P450 involved in the formation of blue flower colors[J]. National Acad Sciences, 1999, 2(96): 778-783.
[3]	Sandra E, Graham, Julian A.How similar are P450s and what can their differences teach us[J]. Archives of Biochemistry and Biophysics, 1999, 1(369): 24-29.
[4]	Werck-Reichhart D, Feyereisen R.Cytochromes P450: a success story[J]. Genome Biology, 2000, 6(1): 30031-30039.
[5]	Schuler M A, Werck-Reichhart D.Functional genomics of P450s[J]. Annual Review of Plant Biology, 2003, 1(54): 629-667.
[6]	Soobrattee M A, Neergheen V.Phenolics as potential antioxidant therapeutic agents: mechanism and actions[J]. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 2005, 1(597): 200-213.
[7]	Schoenbohm C, Martens S, Eder C, et al.Identification of the Arabidopsis thaliana flavonoid 3′-hydroxylase gene and functional expression of the encoded P450 enzyme[J]. Biology Chemistry, 2000, 8(381): 749-753.
[8]	刘海峰, 杨成君, 赵权, 等. 山葡萄中类黄酮3'-羟化酶基因(F3'H) cDNA的克隆和分析[J]. 植物生理学通讯, 2009, 45(12): 1186-1190.
[9]	黄文坤, 程红梅, 郭建英, 等. 紫茎泽兰类黄酮F3'H羟化酶基因的克隆、序列分析和原核表达[J]. 植物生理学通讯, 2007, 43(5) : 821-826.
[10]	Toda K, Akasaka M, Dubouzet E G, et al.Structure of flavonoid 3′-hydroxylase gene for pubescence color in soybean[J]. Crop Science, 2005, 6(45): 2212-2217.
[11]	Han Y, Vimolmangkang S, Soria-Guerra RE, et al.Ectopic expression of apple F3′H genes contributes to anthocyanin accumulation in the Arabidopsis tt7 mutant grown under N stress[J]. Plant Physiology, 2010, 2(153): 806-820.
[12]	Katiyar S, Mukhtar H.Tea in chemoprevention of cancer[J]. International Journal of Oncology, 1996, 2(8): 221-238.
[13]	Hodgson J M.Tea flavonoids and cardiovascular disease[J]. Asia Pacific Journal Clinical Nutrition, 2008, 17(suppl1): 288-290.
[14]	Wolfram S, Wang Y, Thielecke F, et al.Anti-obesity effects of green tea: from bedside to bench[J]. Molecular Nutrition & Food Research, 2006, 50(2): 176-187.
[15]	Schaefer H M, Schaefer V, Levey D J, et al.How plant-animal interactions signal new insights in communication[J]. Trends in Ecology & Evolution, 2004, 19(11): 577-584.
[16]	Regan B C, Julliot C, Simmen B, et al.Fruits, foliage and the evolution of primate colour vision[J]. Philosophical Transactions of the Royal Society B: Biolgical Sciences, 2001, 356(1407): 229-283.
[17]	Castellarin S, Gaspero G, Marconi R, et al.Colour variation in red grapevines (Vitis vinifera L.): genomic organisation, expression of flavonoid 3'-hydroxylase, flavonoid 3', 5'-hydroxylase genes and related metabolite profiling of red cyanidin-/blue delphinidin-based anthocyanins in berry skin[J]. BMC Genomics, 2006, 7(12):1471-2164
[18]	Wu Z J, Li X H, Liu Z W, et al.De novo assembly and transcriptome characterization: novel insights into catechins biosynthesis in Camellia sinensis[J]. BMC Plant Biology, 2014, 14(277): 1471-2229.
[19]	Tamura K, Peterson D, Peterson N, et al.MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods[J]. Molecular Biology & Evolution, 2011, 28(10): 2731-2739.
[20]	Wu Z J, Tian C, Jiang Q.Selection of suitable reference genes for qRT-PCR normalization during leaf development and hormonal stimuli in tea plant (Camellia sinensis)[J]. Scientific Reports, 2016: 1-10. DOI: 1038/srep19748.
[21]	Schäffer A, Aravind L, Madden T, et al.Improving the accuracy of PSI-BLAST protein database searches with composition-based statistics and other refinements[J]. Nucleic Acids Research, 2001, 14(29): 2994-3005.
[22]	Zabala G, Vodkin L.Cloning of the pleiotropic T locus in soybean and two recessive alleles that differentially affect structure and expression of the encoded flavonoid 3′ hydroxylase[J]. Genetics, 2003, 1(163): 295-309.
[23]	Gasteiger E, Gattiker A, Hoogland C, et al.EXPASY: the proteomics server for in-depth protein knowledge and analysis[J]. Nucleic Acids Research, 2003, 13(31): 3784-3788.
[24]	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.
[25]	Zhou T S, Zhou R, Yu Y B, et al.Cloning and characterization of a flavonoid 3′-hydroxylase gene from tea plant (Camellia sinensis)[J]. International Journal of Molecular Sciences, 2016, 17(2): 261.
[26]	贺丽虹, 赵淑娟, 胡之璧. 植物细胞色素P450基因与功能研究进展[J]. 药物生物技术, 2008, 15(2): 142-147.
[27]	Chapple C.Molecular-genetic analysis of plant cytochrome p450 dependent monooxygenases[J]. Annual Review of Plant Biology, 1998, 49(1): 311-342.
[28]	Rupasinghe S, Schuler MA.Homology modeling of plant cytochrome P450s[J]. Phytochemistry Reviews, 2006, 5(2/3): 473-505.
[29]	Wei K, Wang L, Zhang C, et al.Transcriptome analysis reveals key flavonoid 3′-hydroxylase and flavonoid 3′, 5′-hydroxylase genes in affecting the ratio of dihydroxylated to trihydroxylated catechins in Camellia sinensis[J]. PLoS One, 2015,10(9): e137925.
[30]	Jiang X, Liu Y J, Li W W, et al.Tissue-specific, development-dependent phenolic compounds accumulation profile and gene expression pattern in tea plant (Camellia sinensis)[J]. PLoS One, 2013, 8(4): e62315.
[31]	岳川, 曹红利, 曾建明, 等. 茶树中植物激素研究进展[J]. 茶叶科学, 2012, 32(5): 382-392.