杏香气形成特异基因PaCCD1的克隆与功能分析

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

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c DNA序列,命名为Pa CCD1。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 k D。氨基酸序列比对发现,Pa CCD1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,Pa CCD1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,Pa CCD1与At CCD1(拟南芥)及碧桃等的CCD1在同一分支上,其中与Pp CCD1(碧桃)和Cm CCD1(甜瓜)的亲缘关系最近。实时荧光定量PCR结果表明,Pa CCD1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Pa CCD1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,Pa CCD1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Pa CCD1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。

A c DNA encoding Pa CCD1 was cloned from the fruit of‘Luntai Xiaobaixing＇apricot based on RNA-sequence by rapid-amplification of c DNA ends（RACE）. The complete c DNA of Pa CCD1 was 1 885 bp. Sequences analysis indicated that the opening reading frame of Pa CCD1 was 1 644 bp and it encoded 547 amino acids. The molecular weight of Pa CCD1 protein is 61.699 k D. Amino acid sequence alignment showed that Pa CCD1 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 Pa CCD1 protein had many glycosylation and phosphorylation sites. Phylogenetic analysis indicated that Pa CCD1 protein was at the same branch of the tree with At CCD1 protein,and the genetic relationship is the closest with Pp CCD1（Prunus persica）and Cm CCD1（melon）. Real time PCR analysis showed that Pa CCD1 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 Pa CCD1 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 Pa CCD1 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 Pa CCD1 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 Pa CCD1 may be the key gene controlling apocarotenoid aroma volatiles formation in apricot fruit.