桂花OfF3'H基因的克隆与表达分析  

Cloning and Expression Analysis of OfF3'H Gene in Osmanthus fragrans

作　　者：鲜小林 潘远智[1] 陈睿[2] 宋海岩[2] Xian Xiaolin;Pan Yuanzhi;Chen Rui;Song Haiyan(College of Landscape Architecture, Sichuan Agricultural University, Chengdu, 611130;Horticulture Institute, Sichuan Academy of Agricultural Sciences & Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (Southwest Region), Ministry of Agriculture, Chengdu, 610066)

机构地区：[1]四川农业大学风景园林学院,成都611130 [2]四川省农业科学院园艺研究所农业部西南地区园艺作物生物学与种质创制重点实验室,成都610066

出　　处：《分子植物育种》2019年第7期2094-2100,共7页Molecular Plant Breeding

基　　金：四川省财政专项资金项目(2016CYTS-010)资助

摘　　要：类黄酮3'-羟化酶(F3'H)属于细胞色素P450单加氧酶家族,是苯丙烷类代谢途径关键成员,在植物色泽形成及类黄酮成分调控方面起重要作用。本研究以四季桂‘金玉台阁’为实验材料,采用cDNA末端快速扩增法(RACE)从盛花末期小花cDNA中克隆了桂花OfF3'H基因(GenBank编号:MH509392)。序列分析结果显示该基因全长为1 856 bp,其中5'端非翻译区长度为102 bp,3'端非翻译区长度为191 bp,开放阅读框长度为1 563 bp,编码由520个氨基酸残基组成的蛋白质。系统进化分析表明Of F3'H含有植物F3'H蛋白典型的保守结构域,且与猕猴桃的AcF3'H蛋白亲缘关系最近。桂花花期不同阶段生理与分子研究发现初花期OfF3'H基因表达量和总黄酮含量均为最低,而花谢期OfF3'H基因表达量和总黄酮含量达到最高。此外,Of F3'H存在10个潜在的磷酸化位点,其中第83位点的丝氨酸和第304位点的苏氨酸在不同植物间高度保守。本研究为深入研究桂花花色形成及植物类黄酮合成调控的分子机制提供一定的理论依据。Flavonoid 3-hydroxyIase (F3'H) belongs to the family of cytochrome P450 monooxygenases, which is a key regulator of phenylpropanoid metabolic pathway and plays a crucial role in regulation of plant color formation and flavonoid components. In the present study, a full-length cDNA encoding flavonoid 3'-hydroxylase, named OJY3'H (GenBank accession: MH509392), was cloned from the tenninal flower cDNA of Osmanthus fragrans cv 'Jin Yu Tai Ge' using rapid amplification of cDNA ends (RACE) method. Sequence analysis showed that OfF3'H was 1 856 bp in length, including 102 bp 5'UTR, 191 bp 3'UTR, 1 563 bp open reading frame, and it encoded a protein composed of 520 amino acid residues. Phylogenetic analysis indicated that OfF3'H contained the typical conserved motifs of plant F3'H, and had the closest phylogenetic relationships with AcF3'H from Actinidia chinensis. Physiological and molecular studies at different flower stages of Osmanthus fragrans indicated the relative expression level of OfF3'H and the content of total flavonoids were the lowest at the early flowering stage, while they reached the highest at the end of flowering stage. Additionally, ten putative phosphorylation sites were detected in OfF3'H, and Ser83, as well as Thr304, was high conserved among plants. The present study could provide a theoretical basis not only for uncovering the molecular mechanism of Osmanthus fragrans color formation but also for revealing the molecular regulation of flavonoid biosynthesis in plant.

关 键 词：桂花 类黄酮3’-羟化酶 荧光定量PCR 总黄酮含量 磷酸化修饰 

分 类 号：S685.13[农业科学—观赏园艺]