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园艺学报 ›› 2018, Vol. 45 ›› Issue (3): 471-482.doi: 10.16420/j.issn.0513-353x.2017-0416

• 研究论文 • 上一篇    下一篇

杏香气形成特异基因PaCCD1的克隆与功能分析

刘盛雨1,章世奎2,卢娟芳1,李文慧2,席万鹏1,3,*   

  1. (1西南大学园艺园林学院,重庆 400716;2新疆农业科学院轮台国家果树资源圃,新疆轮台 841600;3南方山地园艺学教育部重点实验室,重庆 400715)
  • 出版日期:2018-03-25 发布日期:2018-03-25

Molecular Cloning and Function Analysis of Carotenoid Cleavage Dioxygenase 1(CCD1)in Apricot Fruit

LIU Shengyu1,ZHANG Shikui2,LU Juanfang1,LI Wenhui2,and XI Wanpeng1,3,*   

  1. (1College of Horticulture and Landscape Architecture,Southwest University,Chongqing 400716,China;2Agriculture National Fruit Tree Germplasm Repository,Xinjiang Academy of Agricultural Sciences,Luntai,Xinjiang 841600,China;3Key Laboratory of Horticulture Science for Southern Mountainous Regions,Ministry of Education,Chongqing 400715,China)
  • Online:2018-03-25 Published:2018-03-25

摘要: 以‘轮台小白杏’果实为材料,利用转录组测序筛选获得的类胡萝卜素裂解双加氧酶CCD1基因片段设计特异引物,使用 RACE技术克隆全长cDNA序列,命名为PaCCD1。该基因长为1 885 bp,开放阅读框(ORF)1 644 bp,编码547个氨基酸残基,蛋白质分子量为61.699 kD。氨基酸序列比对发现,PaCCD1与甜瓜等其他已知功能的CCD1相似,具有4个保守的组氨酸残基、2个半保守的谷氨酸残基和1个天冬氨酸残基。活性位点分析表明,PaCCD1编码的蛋白存在多个糖基化位点和磷酸化位点。进化树分析表明,PaCCD1与AtCCD1(拟南芥)及碧桃等的CCD1在同一分支上,其中与PpCCD1(碧桃)和CmCCD1(甜瓜)的亲缘关系最近。实时荧光定量PCR结果表明,PaCCD1在杏幼果中表达量最高,在花中表达量次之,而在根、叶和1年生枝条中不表达,具有组织表达特异性。杏果实发育和成熟过程中,果皮和果肉中PaCCD1的表达均显著上调,主要类胡萝卜素β–胡萝卜素和叶黄素的含量显著下降,而脱辅基类胡萝卜素类香气物质二氢–β–紫罗兰酮、β–紫罗兰酮和β–大马酮的含量显著增加。将目的基因通过转化大肠杆菌,获得了体外表达蛋白,饲喂类胡萝卜素的底物特异性测定发现,PaCCD1蛋白能较快吸收β–胡萝卜素产生二氢–β–紫罗兰酮和β–紫罗兰酮,能吸收叶黄质产生β–大马酮。据此推测PaCCD1是控制杏果实脱辅基类胡萝卜素类香气物质形成的关键基因。

关键词: 杏, 脱辅基类胡萝卜素, 香气, CCD, 克隆, 基因表达

Abstract: A cDNA encoding PaCCD1 was cloned from the fruit of‘Luntai Xiaobaixing’apricot based on RNA-sequence by rapid-amplification of cDNA ends(RACE). The complete cDNA of PaCCD1 was 1 885 bp. Sequences analysis indicated that the opening reading frame of PaCCD1 was 1 644 bp and it encoded 547 amino acids. The molecular weight of PaCCD1 protein is 61.699 kD. Amino acid sequence alignment showed that PaCCD1 was similar to other CCD1 proteins. They all shared typical conserved domains including four conserved histidine residues,two semi-conserved glutamate residues and one semi-conserved aspartate residues. Bioactive site analysis showed that PaCCD1 protein had many glycosylation and phosphorylation sites. Phylogenetic analysis indicated that PaCCD1 protein was at the same branch of the tree with AtCCD1 protein,and the genetic relationship is the closest with PpCCD1(Prunus persica)and CmCCD1(melon). Real time PCR analysis showed that PaCCD1 expressed the highest in fruitlets,next in flower,but has no expression in roots,leaves and one-year branch,exhibiting a tissue-specific expression. Additionally,the expression levels of PaCCD1 in peels and pulps were remarkably up-regulated during fruit development and ripening,the content of the main carotenoids in apricot fruit,β-carotene and lutein,presented a significant decrease during this process,while the contents of the apocarotenoid aroma compounds,dihydro-β-ionone,β-ionone and β-damascenone,increased significantly. A PaCCD1 expression protein in vitro was acquired by transforming the target gene in E. coli. Feeding the protein with different carotenoids as specific substrates showed that the PaCCD1 protein can absorb β-carotene,and produce dihydro-β-ionone and β-ionone quickly,also can absorb lutein and produce β-damascenone. Based on these results,we inferred that PaCCD1 may be the key gene controlling apocarotenoid aroma volatiles formation in apricot fruit.

Key words: apricot, apocarotenoid, aroma, carotenoid cleavage dioxygenase, molecular cloning, gene function

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