橄榄类黄酮3'-羟化酶CaF3'H基因的克隆及其表达特性分析

Molecular Cloning and Expression Analysis of Flavonoids 3'-hydroxylase (CaF3'H) in Canarium album

以‘清榄一号’橄榄(Canarium album)果肉为材料,通过RACE技术克隆得到橄榄类黄酮3'-羟化酶(F3'H)cDNA序列全长,命名为CaF3'H(GenBank登录号KY189088.1)。CaF3'H全长为1 649 bp,包含一个1 554 bp开放阅读框(ORF),编码517个氨基酸,5'和3'非编码区(UTR)长度分别为40 bp和55 bp,编码的氨基酸序列含有P450结构域和半胱氨酸亚铁血红素结合域保守区。氨基酸多重比较分析表明:橄榄和芒果的氨基酸序列相似性为78.89%,较之于其他物种相对最高。蛋白结构预测结果显示,CaF3'H蛋白呈弱亲水性,存在跨膜结构域,没有信号肽。实时荧光定量PCR分析显示,CaF3'H在‘清榄一号’和‘檀香’2个橄榄栽培品种的表达量在花后50 d达到最大值,此后其表达量随果实的成熟呈现出波动,总体表达量比花后50 d都低,这与橄榄生长发育时期多酚含量的变化趋势相符,推测CaF3'H基因在调控果实多酚含量过程中发挥着重要作用。

In this study, a Flavonoids 3＇-hydroxylase gene was isolated by RACE from fruit flesh of Chinese olive（Canarium album） cultivar‘Qinglanyihao＇, and designed as CaF3＇H（GenBank accession no. KY189088.1）. The full-length of CaF3＇H cDNA sequence was 1 649 bp in nucleotide, containing an open reading frame of 1 554 bp,predicting encoding for a polypeptide of 517 amino acids, the 5＇ and 3＇ UTR was 40 and 55 bp, respectively. The predicted amino acids contains cytochrome P450 domain and cysteine heme binding region. The multiple alignment of amino acids showed that the CaF3＇H shares 78.89% similarity with F3＇H protein Mangifera indica, greater similarity than other relative plants. Protein structure predicted show that CaF3＇H is a weak hydrophilic protein,with a transmembrane region and no signal-peptid. Quantitative RT-PCR analysis showed that, the transcription level of CaF3＇H Chinese olive cultivars ＇Qinglanyihao＇ and ＇Tanxiang＇ reached a maximum at 50 d after flowering,then displayed fluctuation during the period of fruit development, lower than 50 d after flowering, this transcript fluctuation trend followed the same trend as polyphenol content changed during fruit development. All the results suggested that CaF3＇H play an important role in regulate polyphenol content during fruit development.