万寿菊类胡萝卜素裂解双加氧酶基因CCD1克隆与表达分析

doi:10.3864/j.issn.0578-1752.2017.10.018

摘要/Abstract

摘要： 【目的】克隆万寿菊（Tagetes erecta L.‘Scarletade’）类胡萝卜素裂解双加氧酶基因CCD1（TeCCD1），分析其序列特征和表达特性，为阐明其在类胡萝卜素降解途径中生物学功能及进一步探讨万寿菊花色形成机理提供理论基础。【方法】依据万寿菊花蕾转录组数据，利用同源序列比对结果设计引物，结合RT-PCR技术克隆获得万寿菊CCD1 cDNA全长，分析其序列特征；利用Real-time PCR分析舌状花未开花蕾、半开花蕾、开放的头状花序和完全开放的头状花序4个不同发育时期的基因表达特性。【结果】克隆获得万寿菊CCD1（GenBank登录号：KX557488）的cDNA全长序列为1 746 bp，编码区长度1 626 bp，编码541个氨基酸。蛋白质分析表明TeCCD1为不稳定蛋白，不含信号肽，属RPE65超家族（登录号：PF03055），包含CCD家族保守结构域，主要定位于细胞质。万寿菊CCD1核酸序列与除虫菊CCD1同源性最高，为89%；氨基酸序列分析表明万寿菊CCD1与除虫菊CCD1同源性高达93%，与其他19个不同种属的CCD1同源性在75%—83%，说明TeCCD1是高度保守的基因；系统进化树分析显示TeCCD1的进化基本符合植物分类学的进化规律，并具有明显的种属特征，万寿菊与菊科同源基因亲缘关系最近。Real-time PCR分析表明TeCCD1在舌状花发育过程中均有表达，随舌状花的开放逐渐升高，S4期达到最大值。【结论】克隆获得万寿菊舌状花的CCD1，是典型的CCD家族成员，为高度保守的基因，主要定位于细胞质，万寿菊舌状花颜色变浅可能与CCD1表达量增加导致类胡萝卜素降解有关。

关键词: 万寿菊, 类胡萝卜素裂解双加氧酶基因, 花色, 基因表达

Abstract: 【Objective】Carotenoid Cleavage Dioxygenase 1 gene of Tagetes erecta L. ‘Scarletade’(TeCCD1) was cloned for bioinformatics and gene expression analysis, which can help clarifying its biological functions in carotenoid degradation pathway and providing a theoretical foundation to further clarify the mechanism of African marigold flower color formation.【Method】According to the transcriptome of African marigold flower bud, the full-length cDNA of TeCCD1 had been obtained, and gene expression profile of ray florets at developmental stages of closed bud, semi-open bud, open flower and fully open flower was studied by Real-time PCR.【Result】The full-length sequence of CCD1 cDNA obtained from African marigold is 1 746 bp (GenBank accession number: KX557488), with a coding region length of 1 626 bp, putatively encoding 541 amino acids. Protein analysis indicated that TeCCD1 is an unstable protein and has no signal peptide, which belongs to the RPE65 superfamily (GenBank accession number is PF03055) having the same conserved domain of CCD family, and it is mainly located in the cytoplasm. CCD1 nucleic acid sequence of African marigold is 89% homologous to that of Pyrethrum. Amino acid sequence analysis suggested that CCD1 of African marigold is 93% homologous to that of Pyrethrum, and 75%-83% homologous to that of 19 different species, indicating that TeCCD1 is highly conserved gene. Phylogenetic analysis showed that the evolution of TeCCD1 is basically in accordance with the evolution law of plant taxonomy and has obvious characteristics of species, which has a closest relationship with that of the species in Compositae. The results of Real-time PCR demonstrated that expression of TeCCD1 increased along with the development of ray floretsand reached the maximum value at S4 stage.【Conclusion】CCD1 homolog was cloned in Tagetes erecta L. ‘Scarletade’ identified to be a typical member of the CCD family, which is a highly conserved gene located in the cytoplasm. The color fading of ray florets during the late development phase is possibly caused by the increase of expression of TeCCD1, which contributes to a decrease in carotenoid content.

Key words: Tagetes erecta L., carotenoid cleavage dioxygenase 1 (CCD1), flower color, gene expression

刘晓丛，曾丽，刘国锋，彭勇政，陶懿伟，张邀月，王梦茹. 万寿菊类胡萝卜素裂解双加氧酶基因CCD1克隆与表达分析[J]. 中国农业科学, 2017, 50(10): 1930-1940.

LIU XiaoCong, ZENG Li, LIU GuoFeng, PENG YongZheng, TAO YiWei, ZHANG YaoYue, WANG MengRu. Cloning and Expression Analysis of Carotenoid Cleavage Dioxygenase 1 (CCD1) Gene in Tagetes erecta L.

[J]. Scientia Agricultura Sinica, 2017, 50(10): 1930-1940.

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