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。
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.
关键词
茶树 /
激素处理 /
进化树分析 /
类黄酮3′-羟化酶; /
组织表达
Key words
flavonoid 3′-hydroxylase /
hormonal treatments /
phylogenetic tree /
tissue expression /
Camellia sinensis
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